Treating anemia in subjects by administration of plasmids encoding growth hormone releasing hormone

ABSTRACT

The present invention pertains to compositions and methods for plasmid-mediated supplementation. The compositions and method are useful for retarding the growth of the tumor, and retarding cachexia, wasting, anemia and other effects that are commonly associated in cancer bearing animals. Overall, the embodiments of the invention can be accomplished by delivering an effective amount of a nucleic acid expression construct that encodes a GHRH or functional biological equivalent thereof into a tissue of an animal and allowing expression of the encoded. gene in the animal. For example, when such a nucleic acid sequence is delivered into the specific cells of the tissue specific constitutive expression is achieved.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/339,610 entitled “Plasmid Mediated Treatment for Anemia,Wasting, Immune Dysfunction and Life Extension for the Chronically Ill,”filed on Dec. 11, 2001, the entire content of which is herebyincorporated by reference.

BACKGROUND

The present invention pertains to compositions and methods forplasmid-mediated supplementation. The present invention pertains tocompositions and methods that are useful for retarding the growth rateof abnormal cells, tumor progression reduction, prevention of kidneyfailure, reduction in metastasis, increased survival and otherconditions commonly associated with cancer-bearing animals. Someembodiments of the invention can be accomplished by delivering aneffective amount of a nucleic acid expression construct that encodes aGHRH or functional biological equivalent thereof into a tissue of asubject and allowing expression of the encoded gene in the subject. Forexample, when such a nucleic acid sequence is delivered into thespecific cells of the subject tissue specific constitutive expression isachieved. Furthermore, external regulation of the GHRH or functionalbiological equivalent thereof gene can be accomplished by utilizinginducible promoters that are regulated by molecular switch molecules,which are given to the subject. The preferred method to deliver theconstitutive or inducible nucleic acid encoding sequences of GHRH or thefunctional biological equivalents thereof is directly into the cells ofthe subject by the process of in vivo electroporation. In addition, atreatment for retarding tumor growth, and retarding cachexia or thewasting effects that are commonly associated with tumors is achieved bythe delivery of recombinant GHRH or biological equivalent into thesubject. Anemia, wasting, tumor growth, immune dysfunction, kidneyfailure, cancer, decreased life expectancy, and other conditions can berelated to a specific cancer, tumor, disease or the effects of a diseasetreatment. This invention relates to a plasmid-mediated supplementationfor:

-   1) treating anemia in a subject;-   2) increasing total red blood cell mass in a subject;-   3) decreasing tumor growth in a tumor bearing individual;-   4) preventing or reversing the wasting of a subject;-   5) reversing abnormal weight loss in a subject;-   6) treating immune dysfunction;-   7) preventing the onset of kidney failure-   8) preventing the onset and/or development of metastasis-   9) reversing the suppression of lymphopoesis in a subject; and/or-   10) extending life expectancy and increasing survival for the    chronically ill subject.

The present invention pertains to compositions and methods that areuseful for retarding the growth rate of abnormal cells, tumorprogression reduction, prevention of kidney failure, reduction ofmetastasis, and increased survival in cancer-bearing animals. Overall,the embodiments of the invention can be accomplished by delivering aneffective amount of a nucleic acid expression construct that encodes aGHRH or functional biological equivalent thereof into a tissue of asubject and allowing expression of the encoded gene in the subject. Forexample, when such a nucleic acid sequence is delivered into thespecific cells of the subject tissue specific constitutive expression isachieved. Furthermore, external regulation of the GHRH or functionalbiological equivalent thereof gene can be accomplished by utilizinginducible promoters that are regulated by molecular switch molecules,which are given to the subject. The preferred method to deliver theconstitutive or inducible nucleic acid encoding sequences of GHRH or thefunctional biological equivalents thereof is directly into the cells ofthe subject by the process of in vivo electroporation. In addition, atreatment for retarding the growth of abnormal cells and tumor growth isachieved by the delivery of recombinant GHRH or biological equivalentinto the subject. Anemia, wasting, tumor growth, immune dysfunction,kidney failure, cancer, decreased life expectancy, and other conditionsalso can be related to a specific cancer, tumor, disease or the effectsof a disease treatment GHRH could be also delivered directly, asprotein, by intravenous, subcutaneous or intranasal administration orthrough a slow release pump.

Anemia: Anemia refers to a condition in which there is a reduction ofthe number or volume of red blood corpuscles or of the total amount ofhemoglobin in the bloodstream, resulting in paleness, generalizedweakness, etc. of the subject. The production of red blood cells inmammals is known as erythropoiesis. Erythropoiesis is primarilycontrolled by erythropoietin (“EPO”), an acidic glycoprotein. The EPOstimulates the production of new erythrocytes to replace those lost tothe aging process. Additionally, EPO production is stimulated underconditions of hypoxia, wherein the oxygen supply to the tissues isreduced below normal physiological levels despite adequate perfusion ofthe tissue by blood. Hypoxia may be caused by hemorrhaging,radiation-induced erythrocyte destruction, various anemia's, highaltitude, or long periods of unconsciousness. In response to tissuesundergoing hypoxic stress, EPO will increase red blood cell productionby stimulating the conversion of primitive precursor cells in the bonemarrow into proerythroblasts that subsequently mature, synthesizehemoglobin and are released into the circulation as red blood cells.

EPO is normally present in low concentrations in plasma, where it issufficient to maintain equilibrium between normal blood cell loss (i.e.,through aging) and red blood cell production. Anemia is a decrease inred blood cell mass caused by decreased production or increaseddestruction of red blood cells. EPO supplementation is currently usedfor treatment of the anemia's associated with different diseases, asend-stage renal failure (Cremagnani et al., 1993; Diez et al., 1996) andacquired immunodeficiency syndrome (“AIDS”) (Sowade et al., 1998),particularly in subjects who are being treated with zidovudine (“AZT”).EPO is also used for amelioration of the anemia associated with cancerchemotherapy (Vansteenkiste et al., 2002).

Another group of anemic disorders, each of which results from aninherited abnormality in globin production, is termed thehemoglobinopathies. Hemoglobinopathies include a spectrum of disordersthat can be classified broadly into two types. The first types are thosethat result from an inherited structural alteration in one of the globinchains, for example sickle cell anemia. These disorders give rise to theproduction of abnormal hemoglobin molecules (Papassotiriou et al.,2000). The second major subdivision of hemoglobinopathies, thethalassemias, results from inherited defects in the rate of synthesis ofone or more of the globin chains. This causes ineffectiveerythropoiesis, hemolysis, and varying degrees of anemia due to theinadequate production of red blood cells. Accordingly, EPO can be usedin the treatment of anemia's, for example, hemoglobinopathies that arecharacterized by low or defective red blood cell production and/orincreased red blood cell destruction (Makis et al., 2001; Payen et al.,2001).

Additional prior art has indicated that anemic patients withpanhypopituitarism, a condition in which hemoglobin (“Hb”) concentrationremained as low as 11.0 g/dl in spite of appropriate replacement withthyroid and adrenocortical hormones, were treated with recombinant humangrowth hormone (“GH”) and EPO levels were increased (Sohmiya and Kato,2000). Recombinant human GH was constantly infused subcutaneously for 12months, which caused the plasma erythropoietin (“EPO”) levels to nearlydouble, with a concomitant increase of Hb concentration. When theadministration of human GH was interrupted, both plasma EPO levels andHb concentrations decreased. There was a close correlation betweenplasma GH and EPO levels before and during the human GH administration.Plasma GH levels were well correlated with Hb concentrations before andduring human GH administration. Plasma IGF-I levels were also correlatedwith Hb concentrations, but not with plasma EPO levels.

U.S. Pat. Nos. 5,846,528 (“the '528 patent”) and 6,274,158 (“the '158patent”) teach that conditions of anemia can be treated by deliberatelyincreasing erythropoietin (“EPO”). In addition, the '528 patent teachesthe use of recombinant adeno-associated virus (“AAV”) virions fordelivery of DNA molecules encoding EPO to muscle cells and tissue in thetreatment of anemia. The '528 patent shows a direct in vivo injection ofrecombinant AAV virions into muscle tissue (e.g., by intramuscularinjection), and in vitro transduction of muscle cells that can besubsequently introduced into a subject for treatment. Thus, a sustainedhigh-level expression of a delivered nucleotide sequence encodingerythropoietin results, whereby in vivo secretion from transduced musclecells allows systemic delivery. The '158 patent teaches the use of thesubcutaneous, intravenous or oral administration of recombinant humanEPO as a hemostatic agent for the treatment or prevention of bleedingfrom any organ or body part involved with benign or malignant lesions,surgical traumatic, non-healing/difficult to treat lesions, or radiationinjury.

In brief, anemia can be caused by a specific disease, environmentalfactors, or the effects of a disease treatment. As discussed,circulating levels of EPO can be increased directly (e.g. injections ofrecombinant EPO) or indirectly (e.g. injections of recombinant GH).Although not wanting to be bound by theory, the related art suggeststhat anemic conditions can be successfully treated by methods orcompounds capable of increasing the circulating levels of EPO. However,a skilled artisan recognizes that biological systems are immeasurablycomplex, and the ability to accurately predict what methods or compoundswill elicit a specific biological response is outside the realm of askilled artesian. Only through diligent laboratory experiments caninsight to compounds or methods to treat anemia be discovered.

Wasting: Wasting of a subject can be defined as decreased body weight ofat least 5-10% of the minimum ideal weight of the individual that ischaracterized by significant loss of both adipose tissue and musclemass, which makes weight gain especially difficult for patients with aprogressive disease (e.g. cancer, AIDS etc.). Wasting or cachexia is aclassic clinical phenomenon that evokes historical images of sickbedsand patients with “consumption.” It simply means “poor condition” inGreek. Accelerated loss of skeletal muscle can occur in setting ofcancer, AIDS, or tuberculosis, as well as other chronic conditions(Barber et al., 1999; Weinroth et al., 1995). Weight loss is the mostobvious manifestation of wasting associated with cancer (Nelson, 2000).Other clinical manifestations include anorexia, muscle wasting, and/orloss of adipose tissue and fatigue, which results in poor performancestatus (Davis and Dickerson, 2000). Because weight loss, tumorhistology, and a poor performance status lead to a poor prognosis,wasting can become the direct cause of death. In contrast to simplestarvation, the weight loss cannot be adequately treated with aggressivefeeding. The weight loss therefore cannot be attributed entirely to poorintake, but is also a result of increased basal energy expenditure.

Wasting is present in more than one half of ambulatory cancer patients,and represents a serious problem when treating chronically ill patients.Although not wanting to be bound by theory, cytokine release and/oractivation and liberation of several tumor derived substances ispostulated to be responsible for the wasting syndrome. The related artteaches that many agents have been evaluated for treatment of wasting,with only modest benefit obtained from progestational agents (Barber etal., 1999; Nelson, 2001). In contrast, recombinant growth hormone(“GH”), insulin-like growth factor-I (“IGF-I”) and IGF binding protein 3(“IGFBP-3”) therapies are effective in producing a benefit in cancercachexia (Bartlett et al., 1994). Thus, the related art suggests thatwasting may be treated by methods or compounds that increase thecirculating levels of GH, IGF-I or IGFBP-3. Unfortunately, thecomplexity of biological systems makes it impossible to accuratelypredict what methods or compounds will elicit a specific biologicalresponse. Thus, only through meticulous laboratory experiments can aninsight to useful compounds or methods to treat wasting be elucidated byone skilled in the art.

Cancer and tumor growth: Cancer is one of the leading causes ofmorbidity and mortality in the US and around the world. The averageannual incidence rate for cancer increased in the last 20 years, toreach 475 to 100,000 in 1999. Due to population growth and aging, thenumber of cancer patient is expected to double from 1.3 million to 2.6million between 2000 and 2050. In addition, the number and proportion ofolder persons with cancer are expected to increase dramatically: from389,000 persons aged 75 years and older with newly diagnosedmalignancies in 2000, to 1,102,000 persons in 2050, an increase from 30%to 42% of the cancer population (Edwards et al., 2002). Cancer inelderly has a poor prognosis due to complicating factors as anorexia ofaging, alterations in the gastrointestinal system, the effect ofelevated leptin levels, especially in men, and a variety of changes incentral nervous system neurotransmitters. Body mass declines after theage of about 70 years old. This includes both loss of adipose tissue andmuscle mass. The loss of muscle mass in older individuals is termedsarcopenia. Illness results in an increase of cytokines that produceboth anorexia and cause protein wasting. Many of the causes of cachexiain older individuals are treatable (Morley, 2001; Yeh and Schuster,1999). Tumor growth is accelerated by increases in cytokines and otherpathological changes in cancer patients, but correction of cachexia,anemia, improvement of immune function and a positive nitrogen balancecan decrease tumor growth and its complications (Demetri, 2001; Koo etal., 2001). Thus, a therapy that would address most of thesecomplications could be of important benefit for patients.

Kidney failure: The predicted increase in the number of people withkidney failure and end-stage renal disease places an enormous burden onhealthcare providers system (Hostetter and Lising, 2002). In order toreduce this burden, strategies must be implemented to improve thedetection of kidney disease, and preventative measures must be targetedat those at greatest risk of disease (Crook et al., 2002). Importantrisk factors include hypertension, diabetes, obesity and cancer (AlSuwaidi et al., 2002; Nampoory et al., 2002). Serum creatinine,proteinuria, and microalbuminuria as early detection markers of diseaseare important, but treatments that could delay or prevent kidney failurecould be of significant benefit for patients and the medical system(LeBrun et al., 2000; Sakhuja et al., 2000).

Growth Hormone (“GH”) and Immune Function: The central role of growthhormone (“GH”) is controlling somatic growth in humans and othervertebrates, and the physiologically relevant pathways regulating GHsecretion from the pituitary is well known. The GH production pathway iscomposed of a series of interdependent genes whose products are requiredfor normal growth. The GH pathway genes include: (1) ligands, such as GHand insulin-like growth factor-I (“IGF-I”); (2) transcription factorssuch as prophet of pit 1, or prop 1, and pit 1: (3) agonists andantagonists, such as growth hormone releasing hormone (“GHRH”) andsomatostatin (“SS”), respectively; and (4) receptors, such as GHRHreceptor (“GHRH-R”) and the GH receptor (“GH-R”). These genes areexpressed in different organs and tissues, including the hypothalamus,pituitary, liver, and bone. Effective and regulated expression of the GHpathway is essential for optimal linear growth, as well as homeostasisof carbohydrate, protein, and fat metabolism GH synthesis and secretionfrom the anterior pituitary is stimulated by GHRH and inhibited bysomatostatin, both hypothalamic hormones. GH increases production ofIGF-I, primarily in the liver, and other target organs. IGF-I and GH, inturn, feedback on the hypothalamus and pituitary to inhibit GHRH and GHrelease. GH elicits both direct and indirect actions on peripheraltissues, the indirect effects being mediated mainly by IGF-I.

The immune function is modulated by IGF-I, which has two major effectson B cell development: potentiation and maturation, and as a B-cellproliferation cofactor that works together with interlukin-7 (“IL-7”).These activities were identified through the use of anti-IGF-Iantibodies, antisense sequences to IGF-I, and the use of recombinantIGF-I to substitute for the activity. There is evidence that macrophagesare a rich source of IGF-I. The treatment of mice with recombinant IGF-Iconfirmed these observations as it increased the number of pre-B andmature B cells in bone marrow (Jardieu et al., 1994). The mature B cellremained sensitive to IGF-I as immunoglobulin production was alsostimulated by IGF-I in vitro and in vivo (Robbins et al., 1994).

The production of recombinant proteins in the last 2 decades provided auseful tool for the treatment of many diverse conditions. For example,GH-deficiencies in short stature children, anabolic agent in burn,sepsis, and AIDS patients. However, resistance to GH action has beenreported in malnutrition and infection. Long-term studies on transgenicanimals and in patients undergoing GH therapies have shown nocorrelation in between GH or IGF-I therapy and cancer development. GHreplacement therapy is widely used clinically, with beneficial effects,but therapy is associated several disadvantages: GH must be administeredsubcutaneously or intramuscularly once a day to three times a week formonths, or usually years; insulin resistance and impaired glucosetolerance; accelerated bone epiphysis growth and closure in pediatricpatients (Blethen and MacGillivray, 1997; Blethen and Rundle, 1996).

In contrast, essentially no side effects have been reported forrecombinant GHRH therapies. Extracranially secreted GHRH, as maturepeptide or truncated molecules (as seen with pancreatic islet celltumors and variously located carcinoids) are often biologically activeand can even produce acromegaly (Esch et al., 1982; Thorner et al.,1984). Administration of recombinant GHRH to GH-deficient children oradult humans augments IGF-I levels, increases GH secretionproportionally to the GHRH dose, yet still invokes a response to bolusdoses of recombinant GHRH (Bercu et al., 1997). Thus, GHRHadministration represents a more physiological alternative of increasingsubnormal GH and IGF-I levels (Corpas et al., 1993).

GH is released in a distinctive pulsatile pattern that has profoundimportance for its biological activity (Argente et al., 1996). Secretionof GH is stimulated by the GHRH, and inhibited by somatostatin, and bothhypothalamic hormones (Thorner et al., 1995). GH pulses are a result ofGHRH secretion that is associated with a diminution or withdrawal ofsomatostatin secretion. In addition, the pulse generator mechanism istimed by GH-negative feedback. The endogenous rhythm of GH secretionbecomes entrained to the imposed rhythm of exogenous GH administration.Effective and regulated expression of the GH and insulin-like growthfactor-I (“IGF-I”) pathway is essential for optimal linear growth,homeostasis of carbohydrate, protein, and fat metabolism, and forproviding a positive nitrogen balance (Murray and Shalet, 2000).Numerous studies in humans, sheep or pigs showed that continuousinfusion with recombinant GHRH protein restores the normal GH patternwithout desensitizing GHRH receptors or depleting GH supplies as thissystem is capable of feed-back regulation, which is abolished in the GHtherapies (Dubreuil et al., 1990; Vance, 1990; Vance et al., 1985).Although recombinant GHRH protein therapy entrains and stimulates normalcyclical GH secretion with virtually no side effects, the shorthalf-life of GHRH in vivo requires frequent (one to three times a day)intravenous, subcutaneous or intranasal (requiring 300-fold higher dose)administration. Thus, as a chronic treatment, GHRH administration is notpractical.

Wild type GHRH has a relatively short half-life in the circulatorysystem, both in humans (Frohman et al., 1984) and in farm animals. After60 minutes of incubation in plasma 95% of the GHRH(1-44)NH2 is degraded,while incubation of the shorter (1-40)OH form of the hormone, undersimilar conditions, shows only a 77% degradation of the peptide after 60minutes of incubation (Frohman et al., 1989). Incorporation of cDNAcoding for a particular protease-resistant GHRH analog in a therapeuticnucleic acid vector results in a molecule with a longer half-life inserum, increased potency, and provides greater GH release inplasmid-injected animals (Draghia-Akli et al., 1999), hereinincorporated by reference). Mutagenesis via amino acid replacement ofprotease sensitive amino acids prolongs the serum half-life of the GHRHmolecule. Furthermore, the enhancement of biological activity of GHRH isachieved by using super-active analogs that may increase its bindingaffinity to specific receptors (Draghia-Akli et al., 1999).

Extracranially secreted GHRH, as processed protein species GHRH(1-40)hydroxy or GHRH(1-44) amide or even as shorter truncated molecules, arebiological active (Thorner et al., 1984). It has been reported that alow level of GHRH (100 pg/ml) in the blood supply stimulates GHsecretion (Corpas et al., 1993). Direct plasmid DNA gene transfer iscurrently the basis of many emerging nucleic acid therapy strategies andthus does not require viral genes or lipid particles (Aihara andMiyazaki, 1998; Muramatsu et al., 2001). Skeletal muscle is targettissue, because muscle fiber has a long life span and can be transducedby circular DNA plasmids that express over months or years in animmunocompetent host (Davis et al., 1993; Tripathy et al., 1996).Previous reports demonstrated that human GHRH cDNA could be delivered tomuscle by an injectable myogenic expression vector in mice where ittransiently stimulated GH secretion to a modes extent over a period oftwo weeks (Draghia-Akli et al., 1997).

Administering novel GHRH analog proteins (U.S. Pat. Nos. 5,847,066;5846,936; 5,792,747; 5,776,901; 5,696,089; 5,486,505; 5,137,872;5,084,442, 5,036,045; 5,023,322; 4,839,344; 4,410,512, RE33,699) orsynthetic or naturally occurring peptide fragments of GHRH (U.S. Pat.Nos. 4,833,166; 4,228,158; 4,228,156; 4,226,857; 4,224,316; 4,223,021;4,223,020; 4,223, 019) for the purpose of increasing release of growthhormone have been reported. A GHRH analog containing the followingmutations have been reported (U.S. Pat. No. 5,846,936): Tyr at position1 to His; Ala at position 2 to Val, Leu, or others; Asn at position 8 toGln, Ser, or Thr; Gly at position 15 to Ala or Leu; Met at position 27to Nle or Leu; and Ser at position 28 to Asn. The GHRH analog is thesubject of U.S. patent application Ser. No. 09/624,268 (“the '268 patentapplication”), which teaches application of a GHRH analog containingmutations that improve the ability to elicit the release of growthhormone. In addition, the '268 patent application relates to thetreatment of growth deficiencies; the improvement of growth performance;the stimulation of production of growth hormone in an animal at agreater level than that associated with normal growth; and theenhancement of growth utilizing the administration of growth hormonereleasing hormone analog and is herein incorporated by reference.

U.S. Pat. No. 5,061,690 is directed toward increasing both birth weightand milk production by supplying to pregnant female mammals an effectiveamount of human GHRH or one of it analogs for 10-20 days. Application ofthe analogs lasts only throughout the lactation period. However,multiple administrations are presented, and there is no disclosureregarding administration of the growth hormone releasing hormone (orfactor) as a DNA molecule, such as with plasmid mediated therapeutictechniques.

U.S. Pat. Nos. 5,134,120 (“the '120 patent”) and 5,292,721 (“the '721patent”) teach that by deliberately increasing growth hormone in swineduring the last 2 weeks of pregnancy through a 3 week lactation resultedin the newborn piglets having marked enhancement of the ability tomaintain plasma concentrations of glucose and free fatty acids whenfasted after birth. In addition, the 120 and 721 patents teach thattreatment of the sow during lactation results in increased milk fat inthe colostrum and an increased milk yield. These effects are importantin enhancing survivability of newborn pigs and weight gain prior toweaning. However the 120 and 721 patents provide no teachings regardingadministration of the growth hormone releasing hormone as a DNA form.

Gene Delivery and in vivo Expression: Recently, the delivery of specificgenes to somatic tissue in a manner that can correct inborn or acquireddeficiencies and imbalances was proved to be possible (Herzog et al.,2001; Song et al., 2001; Vilquin et al., 2001). Gene-based drug deliveryoffers a number of advantages over the administration of recombinantproteins. These advantages include the conservation of native proteinstructure, improved biological activity, avoidance of systemictoxicities, and avoidance of infectious and toxic impurities. Inaddition, nucleic acid vector therapy allows for prolonged exposure tothe protein in the therapeutic range, because the newly secreted proteinis present continuously in the blood circulation. In a few cases, therelatively low expression levels achieved after simple plasmidinjection, are sufficient to reach physiologically acceptable levels ofbioactivity of secreted peptides (Danko and Wolff, 1994; Tsurumi et al.,1996).

The primary limitation of using recombinant protein is the limitedavailability of protein after each administration. Nucleic acid vectortherapy using injectable DNA plasmid vectors overcomes this, because asingle injection into the patient's skeletal muscle permits physiologicexpression for extensive periods of time (WO 99/05300 and WO 01/06988).Injection of the vectors promotes the production of enzymes and hormonesin animals in a manner that more closely mimics the natural process.Furthermore, among the non-viral techniques for gene transfer in vivo,the direct injection of plasmid DNA into muscle tissue is simple,inexpensive, and safe.

In a plasmid-based expression system, a non-viral gene vector may becomposed of a synthetic gene delivery system in addition to the nucleicacid encoding a therapeutic gene product. In this way, the risksassociated with the use of most viral vectors can be avoided. Thenon-viral expression vector products generally have low toxicity due tothe use of “species-specific” components for gene delivery, whichminimizes the risks of immunogenicity generally associated with viralvectors. Additionally, no integration of plasmid sequences into hostchromosomes has been reported in vivo to date, so that this type ofnucleic acid vector therapy should neither activate oncogenes norinactivate tumor suppressor genes. As episomal systems residing outsidethe chromosomes, plasmids have defined pharmacokinetics and eliminationprofiles, leading to a finite duration of gene expression in targettissues.

Efforts have been made to enhance the delivery of plasmid DNA to cellsby physical means including electroporation, sonoporation, and pressure.Administration by electroporation involves the application of a pulsedelectric field to create transient pores in the cellular membranewithout causing permanent damage to the cell. It thereby allows for theintroduction of exogenous molecules (Smith and Nordstrom, 2000). Byadjusting the electrical pulse generated by an electroporetic system,nucleic acid molecules can travel through passageways or pores in thecell that are created during the procedure. U.S. Pat. No. 5,704,908describes an electroporation apparatus for delivering molecules to cellsat a selected location within a cavity in the body of a patient. Thesepulse voltage injection devices are also described in U.S. Pat. No.5,439,440 and 5,702,304, and PCT WO 96/12520, 96/12006, 95/19805, and97/07826.

Recently, significant progress has been obtained using electroporationto enhance plasmid delivery in vivo. Electroporation has been used verysuccessfully to transfect tumor cells after injection of plasmid (Lucaset al., 2002; Matsubara et al., 2001) or to deliver the anti-tumor drugbleomycin to cutaneous and subcutaneous tumors in humans (Gehl et al.,1998; Heller et al., 1996). Electroporation also has been extensivelyused in mice (Lesbordes et al., 2002; Lucas et al., 2001; Vilquin etal., 2001), rats (Terada et al., 2001; Yasui et al., 2001), and dogs(Fewell et al., 2001) to deliver therapeutic genes that encode for avariety of hormones, cytokines or enzymes. Our previous studies usinggrowth hormone releasing hormone (“GHRH”) showed that plasmid therapywith electroporation is scalable and represents a promising approach toinduce production and regulated secretion of proteins in large animalsand humans (Draghia-Akli et al., 1999; Draghia-Akli et al., 2002).

The ability of electroporation to enhance plasmid uptake into theskeletal muscle has been well documented, as described above. Inaddition, plasmid formulated with poly-L-glutamate (“PLG”) orpolyvinylpyrolidone (PVP) has been observed to increase plasmidtransfection and consequently expression of the desired transgene. Theanionic polymer sodium PLG could enhance plasmid uptake at low plasmidconcentrations, while reducing any possible tissue damage caused by theprocedure. The ability of electroporation to enhance plasmid uptake intothe skeletal muscle has been well documented, as previously described.PLG is a stable compound and resistant to relatively high temperatures(Dolnik et al., 1993). PLG has been previously used to increasestability in vaccine preparations (Matsuo et al., 1994) withoutincreasing their immunogenicity. It also has been used as an anti-toxinpost-antigen inhalation or exposure to ozone (Fryer and Jacoby, 1993).In addition, plasmid formulated with PLG or polyvinylpyrrolidone (PVP)has been observed to increase gene transfection and consequently geneexpression to up to 10 fold in the skeletal muscle of mice, rats anddogs (Fewell et al., 2001; Mumper et al., 1998). PLG has been used toincrease stability of anti-cancer drugs (Li et al., 2000) and as “glue”to close wounds or to prevent bleeding from tissues during wound andtissue repair (Otani et al., 1996; Otani et al., 1998).

Although not wanting to be bound by theory, PLG will increase thetransfection of the plasmid during the electroporation process, not onlyby stabilizing the plasmid DNA, and facilitating the intracellulartransport through the membrane pores, but also through an activemechanism. For example, positively charged surface proteins on the cellscould complex the negatively charged PLG linked to plasmid DNA throughprotein-protein interactions. When an electric field is applied, thesurface proteins reverse direction and actively internalize the DNAmolecules, process that substantially increases the transfectionefficiency. Furthermore, PLG will prevent the muscle damage associatedwith in vivo plasmid delivery (Draghia-Akli et al., 2002a) and willincrease plasmid stability in vitro prior to injection.

The use of directly injectable DNA plasmid vectors has been limited inthe past. The inefficient DNA uptake into muscle fibers after simpledirect injection has led to relatively low expression levels (Prenticeet al., 1994; Wells et al., 1997) In addition, the duration of thetransgene expression has been short (Wolff et al., 1990). The mostsuccessful previous clinical applications have been confined to vaccines(Danko and Wolff, 1994; Tsurumi et al., 1996).

Although there are references in the art directed to electroporation ofeukaryotic cells with linear DNA (McNally et al., 1988; Neumann et al.,1982) (Toneguzzo et al., 1988) (Aratani et al., 1992; Naim et al., 1993;Xie and Tsong, 1993; Yorifuji and Mikawa, 1990), these examplesillustrate transfection into cell suspensions, cell cultures, and thelike, and the transfected cells are not present in a somatic tissue.

U.S. Pat. No. 4,956,288 is directed to methods for preparing recombinanthost cells containing high copy number of a foreign DNA byelectroporating a population of cells in the presence of the foreignDNA, culturing the cells, and killing the cells having a low copy numberof the foreign DNA.

U.S. Pat. Nos. 5,874,534 (“the '534 patent”) and U.S. Pat. No. 5,935,934(“the '934 patent”) describe mutated steroid receptors, methods fortheir use and a molecular switch for nucleic acid vector therapy, theentire content of each is hereby incorporated by reference. A molecularswitch for regulating expression in nucleic acid vector therapy andmethods of employing the molecular switch in humans, animals, transgenicanimals and plants (e.g. GeneSwitch®) are described in the '534 patentand the '934 patent. The molecular switch is described as a method forregulating expression of a heterologous nucleic acid cassette fornucleic acid vector therapy and is comprised of a modified steroidreceptor that includes a natural steroid receptor DNA binding domainattached to a modified ligand binding domain. The modified bindingdomain usually binds only non-natural ligands, anti-hormones ornon-native ligands. One skilled in the art readily recognizes naturalligands do not readily bind the modified ligand-binding domain andconsequently have very little, if any, influence on the regulation orexpression of the gene contained in the nucleic acid cassette.

In summary, treatments for conditions such as anemia, wasting and immunedysfunction are uneconomical and restricted in scope. The related arthas shown that it is possible to treat these different diseaseconditions in a limited capacity utilizing recombinant proteintechnology, but these treatments have some significant drawbacks. It hasalso been taught that nucleic acid expression constructs that encoderecombinant proteins are viable solutions to the problems of frequentinjections and high cost of traditional recombinant therapy. Theintroduction of point mutations into the encoded recombinant proteinswas a significant step forward in producing proteins that are morestable in vivo than the wild type counterparts. Unfortunately, eachamino acid alteration in a given recombinant protein must be evaluatedindividually, because the related art does not teach one skilled in theart to accurately predict how changes in structure (e.g. amino-acidsequences) will lead to changed functions (e.g. increased or decreasedstability) of a recombinant protein. Therefore, the beneficial effectsof nucleic acid expression constructs that encode expressed proteins canonly be ascertained through direct experimentation. There is a need inthe art to expanded treatments for subjects with a disease by utilizingnucleic acid expression constructs that are delivered into a subject andexpress stable therapeutic proteins in vivo.

SUMMARY OF THE INVENTION

The present invention pertains to compositions and methods that areuseful for retarding the growth of abnormal cells, tumor progressionreduction, prevention of kidney failure, reduction of metastasis, andincreased survival in cancer-bearing animals. The method of thisinvention comprises treating a subject with plasmid mediated genesupplementation. The method comprises delivering an effective amount ofa nucleic acid expression construct that encodes agrowth-hormone-releasing-hormone (“GHRH”) or functional biologicalequivalent thereof into a tissue, such as a muscle, of the subject.Specific embodiments of this invention are directed toward various typesof tumors, such as adenoma; carcinoma; leukemia; lymphoma; lung tumor;mast cell tumor; melanoma; sarcoma; and solid tumors. The subsequent invivo expression of the GHRH or biological equivalent in the subject issufficient to retard tumor growth, prevent kidney failure and increasesurvival in cancer-bearing animals. It is also possible to enhance thismethod by placing a plurality of electrodes in a selected tissue, thendelivering nucleic acid expression construct to the selected tissue inan area that interposes the plurality of electrodes, and applying acell-transfecting pulse to the selected tissue in an area of theselected tissue where the nucleic acid expression construct wasdelivered. Electroporation, direct injection, gene gun, or gold particlebombardment are also used in specific embodiments to deliver the nucleicacid expression construct encoding the GHRH or biological equivalentinto the subject. The subject in this invention comprises mammals, suchas a humans, and domesticated animals.

The composition of this invention comprises an effective amount of anucleic acid expression construct that encodes agrowth-hormone-releasing-hormone (“GHRH”) or functional biologicalequivalent thereof, wherein delivering and subsequent expression of theGHRH or biological equivalent in a tissue of the subject is sufficientto retard tumor growth and retard cachexia or the wasting effects thatare commonly associated with tumor growth. Specific elements of thenucleic acid expression construct of this invention are also described.For example, the construct comprises a tissue specific promoter; a GHRHor functional biological equivalent; and a 3′ untranslated region(“3“UTR”) that are operatively linked. The nucleic acid expressionconstruct of this invention comprises a construct that is substantiallyfree of a viral backbone. Specific examples of a nucleic acid expressionconstructs used for this invention are also presented. The encodedfunctional biological equivalent of GHRH comprises a polypeptide havingsimilar or improved biological activity when compared to the GHRHpolypeptide. The GHRH or functional biological equivalent that isencoded by the nucleic acid expression construct and useful for thisinvention comprises an amino acid structure with a general sequence asfollows:-X₁-X₂-DAIFTNSYRKVL-X₃-QLSARKLLQDI-X₄-X₅-RQQGERNQEQGA-OHwherein the formula has the following characteristics: X₁ is a D-orL-isomer of the amino acid tyrosine (“Y”), or histidine (“H”); X₂ is aD-or L-isomer of the amino acid alanine (“A”), valine (“V”), orisoleucine (“I”); X₃ is a D-or L-isomer of the amino acid alanine (“A”)or glycine (“G”); X₄ is a D-or L-isomer of the amino acid methionine(“M”), or leucine (“L”); X₅ is a D-or L-isomer of the amino acid serine(“S”) or asparagine (“N”); or a combination thereof. Specific examplesof amino acid sequences for GHRH or functional biological equivalentsthat are useful for this invention are presented. In a specificembodiment, the encoded GHRH or functional biological equivalent thereoffacilitates growth hormone (“GH”) secretion in a subject that hasreceived the nucleic acid expression construct. In specific embodimentsof the invention, a transfection-facilitating polypeptide that increasethe tissues ability to uptake the nucleic acid expression constructcomprises a charged polypeptide, such as poly-L-glutamate.

One embodiment of the present invention pertains to a plasmid mediatedsupplementation method for treating anemia; increasing total red bloodcell mass in a subject; reversing the wasting; reversing abnormal weightloss; treating immune dysfunction; reversing the suppression oflymphopoesis; or extending life expectancy for the chronically illsubject. This can be achieved utilizing an effective amount of a nucleicacid expression construct that contains both a constitutive promoter andan encoding sequence for growth hormone releasing hormone (“GHRH”) orbiological equivalent thereof. When this nucleic acid sequence isdelivered into the specific cells of the subject (e.g. somatic cells,stem cells, or germ cells), tissue specific and constitutive expressionof GHRH is achieved. The preferred method to deliver the nucleic acidsequence with the constitutive promoter and the encoding sequence ofGHRH or the biological equivalent thereof is directly into the cells ofthe subject by the process of in vivo electroporation. Electroporationmay involve externally supplied electrodes, or in the case of needles,internally supplied electrodes to aid in the inclusion of desirednucleotide sequences into the cells of a subject while the cells arewithin a tissue of the subject.

A further embodiment of the present invention pertains to pertains to aplasmid mediated method for the treatment of anemia, wasting, immunedysfunction and life extension for the chronically ill subject byutilizing the ability to regulate the expression of GHRH or biologicalequivalent thereof. Regulation is achieved by delivering into the cellsof the subject a first nucleic acid sequence, and a second nucleic acidsequence, followed by a molecular switch; where the first nucleic acidsequence contains an inducible-promoter with a coding region for agrowth-hormone-releasing-hormone (“inducible-GHRH”) or an biologicalequivalent thereof and the second nucleic acid sequence has aconstitutive promoter with a coding region for an inactive regulatorprotein. By delivering a molecular switch molecule (e.g. mifepistone)into the subject, the inactive regulator protein becomes active andinitiates transcription of the inducible-GHRH in the subject. Theexternal regulation, expression and ensuing release of GHRH orbiological equivalent thereof by the modified-cells within the subjectwill the conditions of anemia, wasting, immune dysfunction and lifeextension for the chronically ill subject. The delivery of the nucleicacid sequences that allow external regulation of GHRH or the biologicalequivalent thereof directly into the cells of the subject can beaccomplished by the process of in vivo electroporation.

A further embodiment of the present invention pertains to a method oftreatment for anemia, wasting, immune dysfunction and life extension forthe chronically ill subject by utilizing therapy that introducesspecific recombinant GHRH-biological equivalent protein into thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows the amino acid sequence of GHRH or biological equivalentthereof.

FIG. 2 shows the increase in the percentage of IGF-I levels in healthydogs that were injected with different concentrations of the pSP-HV-GHRHplasmid.

FIG. 3 shows the increase in the percentage of IGF-I levels at 9-27 and28-56 days post-injection in dogs with cancer that were injected at day0 with 100 mcg/kg to a total of no more than 1000 mcg of the pSP-HV-GHRHplasmid.

FIG. 4 shows the percentage of weight gain in healthy dogs that wereinjected with different concentrations of the pSP-HV-GHRH plasmid.

FIG. 5 shows the number of red blood cells in dogs with spontaneousmalignancies treated with the GHRH plasmid therapy compared with controldogs with cancer.

FIG. 6 shows hemoglobin values in dogs with spontaneous malignanciestreated with the GHRH plasmid therapy compared with control dogs withcancer.

FIG. 7 shows hematocrit levels in dogs with spontaneous malignanciestreated with the GHRH plasmid therapy compared with control dogs withcancer.

FIG. 8 shows the percentage of lymphocytes in dogs with spontaneousmalignancies treated with the GHRH plasmid therapy compared with controldogs with cancer.

FIG. 9 shows a schematic of the mifepristone-dependent GHRH/GeneSwitch®system. Plasmid pl633 encodes for the GeneSwitch® regulator protein,that is a chimera of yeast GAL4 DNA binding domain (“GAL4”), truncatedhuman progesterone receptor ligand-binding domain (“hPR LBD”), andactivation domain from the p65 subunit of human NF-κB (“p65”). Theprotein is synthesized as an inactive monomer. Binding of mifepristonetriggers a conformational change that leads to activation anddimerization. Activated homodimers bind to GAL4 sites in the induciblepromoter and stimulate transcription of the GHRH gene.

FIG. 10 shows tumor volume progression in immunocompetent C57/B16 micethat received 2×10⁶ Lewis lung adenocarcinoma cells in their left flank.Treated animals received at 1 day after tumor cells implantation 20micrograms of plasmid expressing human growth hormone releasing hormone,while controls received a control beta-galactosidase plasmid. GHRHtreated tumor bearing animals have significantly slower tumordevelopment and progression.

FIG. 11 shows survival time for immunocompetent C57/B16 mice thatreceived 2×10⁶ Lewis lung adenocarcinoma cells in their left flank.Treated animals received at 1 day after tumor cells implantation 20micrograms of plasmid expressing human growth hormone releasing hormone,while controls received a control beta-galactosidase plasmid.GHRH-treated tumor bearing animals have increase survival.

FIG. 12 shows kidney size for immunocompetent C57/B16 mice that received2×10⁶ Lewis lung adenocarcinoma cells in their left flank. Treatedanimals received at 1 day after tumor cells implantation 20 microgramsof plasmid expressing human growth hormone releasing hormone, whilecontrols received a control beta-galactosidase plasmid. Control animalshave significantly smaller kidney size, sign of kidney failure.

FIG. 13 shows that the markers associtaed with metastasis developmentare increased in control animals versus GHRH-treated animals, and thatthe histopathology report showed considerably more metastasis in thecontrol animals than in treated once.

FIG. 14 shows that tumor growth did not increase in nude mice treatedwith plasmid-mediated growth hormone releasing hormone (constitutivelyactive pGHRH or regulated Gene Switch system GHRH-IS+/−MFP). NCI—humanlung adenocarcinoma cell line. Animals treated with the constitutivelyactive GHRH had smaller tumors than controls (p<0.02) at 33 dayspost-treatment. No other group displayed significant differences whencompared to controls.

FIG. 15 shows the protein metabolism in dogs at 56 days post-injection.

FIG. 16 shows the blood values in dogs at 56 days post-injection.

FIG. 17 shows the bone metabolism in dogs at 56 days post-injection.

FIG. 18 shows the diagnosis, specific therapy chart and survival fordogs with spontaneous cancer.

FIG. 19 shows the blood values for dogs with cancer.

FIG. 20 shows the blood values for old healthy dogs.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Definitions

The term “a” or “an” as used herein in the specification may mean one ormore. As used herein in the claim(s), when used in conjunction with theword “comprising”, the words “a” or “an” may mean one or more than one.As used herein “another” may mean at least a second or more.

The term “abnormal weight loss,” as used herein is defined as decreasedbody weight of at least 5-10% of the minimum ideal weight of theindividual that is characterized by significant loss of both adiposetissue and muscle mass.

The term “AIDS therapy” as used herein refers to treatment of acquiredimmune deficiency syndrome (“AIDS”) by any medical or physical means,including, but not limited to: antiretrovirals, nucleoside analogues,non-nucleoside reverse transcriptase inhibitors (NNRTIs), proteaseinhibitors, and/or other drugs used to boost the immune system.

The term “analog” as used herein includes any mutant of GHRH, orsynthetic or naturally occurring peptide fragments of GHRH, such asHV-GHRH (SEQ ID NO: 1). TI-GHRH (SEQ ID NO: 2). TV-GHRH (SEQ ID NO: 3),15127/28-GHRH (SEQ ID NO: 4), (1-44)NH2 (SEQ ID NO: 5). OR (1-40)OH (SEQID NO: 6) forms, or any shorter form to no less than (1-29) amino acids.

The term “anemia” as used herein refers to a condition in which there isa reduction of the number and/or volume of red blood corpuscles or ofthe total amount of hemoglobin in the bloodstream, resulting inpaleness, generalized weakness, etc., of the subject.

The term “antiviral therapy” as used herein refers to a group of drugsthat are of three main types, including: nucleoside analog drugs,protease (proteinase) inhibitor drugs, and non-nucleosidereverse-transcriptase inhibitor drugs (NNRTIs).

The term “bodily fat proportion” as used herein is defined as the bodyfat mass divided by the total body weight.

The term “cancer therapy” as used herein refers to treatment of cancerby any medical or physical means, including, but not limited to surgery,immunotherapy, chemotherapy, radiation therapy, hyperthermia and/orphotodynamic therapy.

The term “cachexia” as used herein is defined as the accelerated loss ofskeletal muscle.

The term “cassette” as used herein is defined as one or more transgeneexpression vectors.

The term “cell-transfecting pulse” as used herein is defined as atransmission of a force which results in transfection of a vector, suchas a linear DNA fragment, into a cell. In some embodiments, the force isfrom electricity, as in electroporation, or the force is from vascularpressure.

The term “chronically ill” as used herein is defined as patients withconditions as chronic obstructive pulmonary disease, chronic heartfailure, stroke, dementia, rehabilitation after hip fracture, chronicrenal failure, rheumatoid arthritis, and multiple disorders in theelderly, with doctor visits and/or hospitalization once a month for atleast two years.

The term “coding region” as used herein refers to any portion of the DNAsequence that is transcribed into messenger RNA (mRNA) and thentranslated into a sequence of amino acids characteristic of a specificpolypeptide.

The term “coding region” as used herein refers to any portion of the DNAsequence that is transcribed into messenger RNA (mRNA) and thentranslated into a sequence of amino acids characteristic of a specificpolypeptide.

The term “delivery” or “delivering” as used herein is defined as a meansof introducing a material into a tissue, a subject, a cell or anyrecipient, by means of chemical or biological process, injection,mixing, electroporation, sonoporation, or combination thereof, eitherunder or without pressure.

The term “DNA fragment” or “nucleic acid expression construct” as usedherein refers to a substantially double stranded DNA molecule. Althoughthe fragment may be generated by any standard molecular biology meansknown in the art, in some embodiments the DNA fragment or expressionconstruct is generated by restriction digestion of a parent DNAmolecule. The terms “expression vector,” “expression cassette,” or“expression plasmid” can also be used interchangeably. Although theparent molecule may be any standard molecular biology DNA reagent, insome embodiments the parent DNA molecule is a plasmid.

The term “donor-subject” as used herein refers to any species of theanimal kingdom wherein cells have been removed and maintained in aviable state for any period of time outside the subject.

The term “donor-cells” as used herein refers to any cells that have beenremoved and maintained in a viable state for any period of time outsidethe donor-subject.

The term “effective amount” as used herein refers to sufficient nucleicacid expression construct or encoded protein administered to humans,animals or into tissue culture to produce the adequate levels ofprotein, RNA, or hormone. One skilled in the art recognizes that theadequate level of protein or RNA will depend on the use of theparticular nucleic acid expression construct. These levels will bedifferent depending on the type of administration and treatment orvaccination.

The term “electroporation” as used herein refers to a method thatutilized electric pulses to deliver a nucleic acid sequence into cells.

The terms “electrical pulse” and “electroporation” as used herein referto the administration of an electrical current to a tissue or cell forthe purpose of taking up a nucleic acid molecule into a cell. A skilledartisan recognizes that these terms are associated with the terms“pulsed electric field” “pulsed current device” and “pulse voltagedevice.” A skilled artisan recognizes that the amount and duration ofthe electrical pulse is dependent on the tissue, size, and overallhealth of the recipient subject, and furthermore knows how to determinesuch parameters empirically.

The term “encoded GHRH” as used herein is a biologically activepolypeptide of growth hormone releasing hormone.

The term “functional biological equivalent” of GHRH as used herein is apolypeptide that has a distinct amino acid sequence from a wild typeGHRH polypeptide while simultaneously having similar or improvedbiological activity when compared to the GHRH polypeptide. Thefunctional biological equivalent may be naturally occurring or it may bemodified by an individual. A skilled artisan recognizes that the similaror improved biological activity as used herein refers to facilitatingand/or releasing growth hormone or other pituitary hormones. A skilledartisan recognizes that in some embodiments the encoded functionalbiological equivalent of GHRH is a polypeptide that has been engineeredto contain a distinct amino acid sequence while simultaneously havingsimilar or improved biological activity when compared to the GHRHpolypeptide. Methods known in the art to engineer such a sequenceinclude site-directed mutagenesis.

The term “growth hormone releasing hormone” (“GHRH”) as used herein isdefined as a hormone that facilitates or stimulates release of growthhormone, and in a lesser extent other pituitary hormones, as prolactin.

The term “growth hormone” (“GH”) as used herein is defined as a hormonethat relates to growth and acts as a chemical messenger to exert itsaction on a target cell.

The term “GeneSwitch®” (a registered trademark of Valentis, Inc.;Burlingame, Calif.) as used herein refers to the technology of amifepristone-inducible heterologous nucleic acid sequences encodingregulator proteins, GHRH, biological equivalent or combination thereof.Such a technology is schematically diagramed in FIG. 1 and FIG. 9. Askilled artisan recognizes that antiprogesterone agent alternatives tomifepristone are available, including onapristone, ZK112993, ZK98734,and 5α pregnane-3,2-dione.

The term “growth hormone” (“GH”) as used herein is defined as a hormonethat relates to growth and acts as a chemical messenger to exert itsaction on a target cell. In a specific embodiment, the growth hormone isreleased by the action of growth hormone releasing hormone.

The term “growth hormone releasing hormone” (“GHRH”) as used herein isdefined as a hormone that facilitates or stimulates release of growthhormone, and in a lesser extent other pituitary hormones, such asprolactin.

The term “heterologous nucleic acid sequence” as used herein is definedas a DNA sequence comprising differing regulatory and expressionelements.

The term “immune dysfunction” as used herein refers to the abnormal,impaired, or incomplete functioning of a subject's immune system, asdetermined indirectly or directly by immune specific markers (e.g. IGF-Ilevels, or % lymphocytes).

The term “immunotherapy” as used herein refers to any treatment thatpromotes or enhances the body's immune system to build protectiveantibodies that will reduce the symptoms of a medical condition and/orlessen the need for medications.

The term “lean body mass” (“LBM”) as used herein is defined as the massof the body of an animal attributed to non-fat tissue such as muscle.

The term “life extension for the chronically ill” as used herein refersto an increase in the actual life expectancy for a subject thatundertakes the treatment compared to a subject that did not havetreatment.

The term “lymphopoiesis” as used herein is defined as the production oflymphocytes.

The term “kidney failure” as used herein is defined as the abrupt orchronic decline in glomerular filtration rate resulting from ischemic ortoxic injury to the kidney, and includes a decrease of glomerularcapillary permeability, back-leak of glomerular filtrate, tubularobstruction, and intrarenal vasoconstriction.

The term “modified cells” as used herein is defined as the cells from asubject that have an additional nucleic acid sequence introduced intothe cell.

The term “modified-donor-cells” as used herein refers to any donor-cellsthat have had a GHRH-encoding nucleic acid sequence delivered.

The term “molecular switch” as used herein refers to a molecule that isdelivered into a subject that can regulate transcription of a gene.

The term “nucleic acid expression construct” as used herein refers toany type of genetic construct comprising a nucleic acid coding for a RNAcapable of being transcribed. The term “expression vector” can also beused interchangeably herein. In specific embodiments, the nucleic acidexpression construct comprises: a promoter; a nucleotide sequence ofinterest; and a 3′ untranslated region; wherein the promoter, thenucleotide sequence of interest, and the 3′ untranslated region areoperatively linked; and in vivo expression of the nucleotide sequence ofinterest is regulated by the promoter.

The term “operatively linked” as used herein refers to elements orstructures in a nucleic acid sequence that are linked by operativeability and not physical location. The elements or structures arecapable of, or characterized by accomplishing a desired operation. It isrecognized by one of ordinary skill in the art that it is not necessaryfor elements or structures in a nucleic acid sequence to be in a tandemor adjacent order to be operatively linked.

The term “poly-L-glutamate (“PLG”)” as used herein refers to abiodegradable polymer of L-glutamic acid that is suitable for use as avector or adjuvant for DNA transfer into cells with or withoutelectroporation.

The term “post-injection” as used herein refers to a time periodfollowing the introduction of a nucleic acid cassette that containsheterologous nucleic acid sequence encoding GHRH or a biologicalequivalent thereof into the cells of the subject and allowing expressionof the encoded gene to occur while the modified cells are within theliving organism.

The term “plasmid” as used herein refers generally to a constructioncomprised of extra-chromosomal genetic material, usually of a circularduplex of DNA that can replicate independently of chromosomal DNA.Plasmids, or fragments thereof, may be used as vectors. Plasmids aredouble-stranded DNA molecule that occur or are derived from bacteria and(rarely) other microorganisms. However, mitochondrial and chloroplastDNA, yeast killer and other cases are commonly excluded.

The term “plasmid mediated gene supplementation” as used herein refers amethod to allow a subject to have prolonged exposure to a therapeuticrange of a therapeutic protein by utilizing an effective amount of anucleic acid expression construct in vivo.

The term “pulse voltage device,” or “pulse voltage injection device” asused herein relates to an apparatus that is capable of causing or causesuptake of nucleic acid molecules into the cells of an organism byemitting a localized pulse of electricity to the cells. The cellmembrane then destabilizes, forming passageways or pores. Conventionaldevices of this type are calibrated to allow one to select or adjust thedesired voltage amplitude and the duration of the pulsed voltage. Theprimary importance of a pulse voltage device is the capability of thedevice to facilitate delivery of compositions of the invention,particularly linear DNA fragments, into the cells of the organism.

The term “plasmid backbone” as used herein refers to a sequence of DNAthat typically contains a bacterial origin of replication, and abacterial antibiotic selection gene, which are necessary for thespecific growth of only the bacteria that are transformed with theproper plasmid. However, there are plasmids, called mini-circles, thatlack both the antibiotic resistance gene and the origin of replication(Darquet et al., 1997; Darquet et al., 1999; Soubrier et al., 1999). Theuse of in vitro amplified expression plasmid DNA (i.e. non-viralexpression systems) avoids the risks associated with viral vectors. Thenon-viral expression systems products generally have low toxicity due tothe use of “species-specific” components for gene delivery, whichminimizes the risks of immunogenicity generally associated with viralvectors. One aspect of the current invention is that the plasmidbackbone does not contain viral nucleotide sequences.

The term “promoter” as used herein refers to a sequence of DNA thatdirects the transcription of a gene. A promoter may direct thetranscription of a prokaryotic or eukaryotic gene. A promoter may be“inducible”, initiating transcription in response to an inducing agentor, in contrast, a promoter may be “constitutive”, whereby an inducingagent does not regulate the rate of transcription. A promoter may beregulated in a tissue-specific or tissue-preferred manner, such that itis only active in transcribing the operable linked coding region in aspecific tissue type or types.

The term “radiation therapy” as used herein refers to radiationtreatment given to cancer patients that damages the DNA in cancer cells,which often results in the death of cancer cells.

The term “replication element” as used herein comprises nucleic acidsequences that will lead to replication of a plasmid in a specifiedhost. One skilled in the art of molecular biology will recognize thatthe replication element may include, but is not limited to a selectablemarker gene promoter, a ribosomal binding site, a selectable marker genesequence, and a origin of replication.

The term “residual linear plasmid backbone” as used herein comprises anyfragment of the plasmid backbone that is left at the end of the processmaking the nucleic acid expression plasmid linear.

The term “recipient-subject” as used herein refers to any species of theanimal kingdom wherein modified-donor-cells can be introduced from adonor-subject.

The term “red blood cell mass” (“RBC-mass”) of a subject as used hereinis determined using one of the three following tests: 1) Hematocrit: thepercentage of red blood cells in plasma; 2) red blood cell (“RBC”)count: the number of red blood cells in plasma; and 3) hemoglobin: thelevel of oxygen-carrying protein within the subjects' red blood cells.

The term “regulator protein” as used herein refers to any protein thatcan be used to control the expression of a gene.

The term “regulator protein” as used herein refers to protein thatincreasing the rate of transcription in response to an inducing agent.

The terms “subject” or “animal” as used herein refers to any species ofthe animal kingdom. In preferred embodiments, it refers morespecifically to humans and domesticated animals used for: pets (e.g.cats, dogs, etc.); work (e.g. horses, etc.); food (cows, chicken, fish,lambs, pigs, etc); and all others known in the art.

The term “tissue” as used herein refers to a collection of similar cellsand the intercellular substances surrounding them. A skilled artisanrecognizes that a tissue is an aggregation of similarly specializedcells for the performance of a particular function. For the scope of thepresent invention, the term tissue does not refer to a cell line, asuspension of cells, or a culture of cells. In a specific embodiment,the tissue is electroporated in vivo. In another embodiment, the tissueis not a plant tissue. A skilled artisan recognizes that there are fourbasic tissues in the body: 1) epithelium; 2) connective tissues,including blood, bone, and cartilage; 3) muscle tissue; and 4) nervetissue. In a specific embodiment, the methods and compositions aredirected to transfer of linear DNA into a muscle tissue byelectroporation.

The term “therapeutic element” as used herein comprises nucleic acidsequences that will lead to an in vivo expression of an encoded geneproduct. One skilled in the art of molecular biology will recognize thatthe therapeutic element may include, but is not limited to a promotersequence, a transgene, a poly A sequence, or a 3′ or 5′ UTR.

The term “transfects” as used herein refers to introduction of a nucleicacid into a eukaryotic cell. In some embodiments, the cell is not aplant tissue or a yeast cell.

The term “vector” as used herein refers to any vehicle that delivers anucleic acid into a cell or organism. Examples include plasmid vectors,viral vectors, liposomes, or cationic lipids.

The term “viral backbone” as used herein refers to a nucleic acidsequence that does not contain a promoter, a gene, and a 3′ poly Asignal or an untranslated region, but contain elements including, butnot limited at site-specific genomic integration Rep and invertedterminal repeats (“ITRs”) or the binding site for the tRNA primer forreverse transcription, or a nucleic acid sequence component that inducesa viral immunogenicity response when inserted in vivo, allowsintegration, affects specificity and activity of tissue specificpromoters, causes transcriptional silencing or poses safety risks to thesubject.

The term “vascular pressure pulse” refers to a pulse of pressure from alarge volume of liquid to facilitate uptake of a vector into a cell. Askilled artisan recognizes that the amount and duration of the vascularpressure pulse is dependent on the tissue, size, and overall health ofthe recipient animal, and furthermore knows how to determine suchparameters empirically.

The term “vector” as used herein refers to a construction comprised ofgenetic material designed to direct transformation of a targeted cell bydelivering a nucleic acid sequence into that cell. A vector may containmultiple genetic elements positionally and sequentially oriented withother necessary elements such that an included nucleic acid cassette canbe transcribed and when necessary translated in the transfected cells.These elements are operatively linked. The term “expression vector”refers to a DNA plasmid that contains all of the information necessaryto produce a recombinant protein in a heterologous cell.

The term “wasting” as used herein is defined as decreased body weightcharacterized by significant loss of both adipose tissue and muscle massthat makes weight gain especially difficult for patients withprogressive diseases, such as cancer or AIDS. Wasting can be related tothe disease itself or the effects of its treatment, or both.

One aspect of the current invention pertains to a method useful forretarding the growth of abnormal cells, and promoteing tumor progressionreduction in cancer-bearing animals. The method of this inventioncomprises treating a subject with plasmid mediated gene supplementation.The method comprises delivering an effective amount of a nucleic acidexpression construct that encodes a growth-hormone-releasing-hormone(“GHRH”) or functional biological equivalent thereof into a tissue, suchas a muscle, of the subject. Specific embodiments of this invention aredirected toward particular types of tumors (e.g. adenoma; carcinoma;leukemia; lymphoma; lung tumor; mast cell tumor; melanoma; sarcoma; andsolid tumors). The subsequent in vivo expression of the GHRH orbiological equivalent in the subject is sufficient to retard tumorgrowth, and retard cachexia or the wasting effects that are commonlyassociated with tumors. It is also possible to enhance this method byplacing a plurality of electrodes in a selected tissue, then deliveringnucleic acid expression construct to the selected tissue in an area thatinterposes the plurality of electrodes, and applying a cell-transfectingpulse (e.g. electrical) to the selected tissue in an area of theselected tissue where the nucleic acid expression construct wasdelivered. However, the cell-transfecting pulse need not be anelectrical pulse, a vascular pressure pulse can also be utilized.Electroporation, direct injection, gene gun, or gold particlebombardment are also used in specific embodiments to deliver the nucleicacid expression construct encoding the GHRH or biological equivalentinto the subject. The subject in this invention comprises an animal(e.g. a human, a pig, a horse, a cow, a mouse, a rat, a monkey, a sheep,a goat, a dog, or a cat).

Specific elements of the nucleic acid expression construct of thisinvention are also described. For example, the construct comprises atissue specific promoter; a GHRH or functional equivalent; and a 3′untranslated region (“3′UTR”) that are operatively linked. In specificembodiments, the tissue-specific promoter comprises a muscle-specificpromoter (e.g., SPc5-12 (SEQ ID NO: 7)), and the 3′ UTR of the nucleicacid expression construct comprises a human growth hormone 3′ UTR (SEQID NO: 8), bovine growth hormone 3′ UTR. skeletal apha actin 3′ UTR orSV40 polyadenylation signal. The nucleic acid expression construct ofthis invention comprises a construct that is substantially free of aviral backbone. Specific examples of a nucleic acid expression constructused for this invention comprises plasmids with SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. The encoded functionalbiological equivalent of GHRH comprises a polypeptide having similar orimproved biological activity when compared to the GHRH polypeptide. TheGHRH or functional biological equivalent that is encoded by the nucleicacid expression construct and useful for this invention comprises anamino acid structure with a general sequence as follows (SEQ ID NO: 6):-X₁-X₂-DAIFTNSYRKVL-X₃-OLSARKLLQDI-X₄-X₅-RQQGERNQEQGA-OHwherein the formula has the following characteristics: X₁ is a D- orL-isomer of the amino acid tyrosine (“Y”), or histidine (“H”); X₂ is aD- or L-isomer of t he amino acid alanine (“A”), valine (“V”), orisoleucine (“I”); X₃ is is D- or L-isomer of the amino acid alanine(“A”) or glycine (“G”); X₄ is a D- or L-isomer of the amino acidmethionine (“M”), or leucine (“L”); X₅ is a D- or L-isomer of the aminoacid serine (“S”) or asparagine (“N”); or a combination thereof.Specific examples of amino acid sequence for GHRH or functionalbiological equivalents that are useful for this invention are presentedin SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; and SEQ ID NO: 10. In aspecific embodiment, the encoded GHRH or functional biologicalequivalent thereof facilitates growth hormone (“GH”) secretion in asubject that has received the nucleic acid expression construct.

Although not wanting to be bound by theory, the ability of cells in atissue to uptake the nucleic acid expression construct can befacilitated by a transfection-facilitating polypeptide. In specificembodiments of the invention, the transfection-facilitating polypeptidecomprises a charged polypeptide such as poly-L-glutamate.

Another aspect of the current invention comprise compositions that areuseful for retarding the growth of abnormal cells, tumor progressionreduction, prevention of kidney failure, reduction of metastasis, andincreased survival in cancer-bearing animals. The composition of thisinvention comprises an effective amount of a nucleic acid expressionconstruct that encodes a growth-hormone-releasing-hormone (“GHRH”) orfunctional biological equivalent thereof, wherein delivering andsubsequent expression of the GHRH or biological equivalent in a tissueof the subject is sufficient to retard the growth of abnormal cells,promote tumor progression reduction, prevent kidney failure, andincrease survival in cancer-bearing animals. Specific embodiments ofthis invention are directed toward particular types of tumors andcancers (e.g. adenoma; carcinoma; leukemia; lymphoma; lung tumor; mastcell tumor; melanoma; sarcoma; and solid tumors).

The subsequent in vivo expression of the GHRH or biological equivalentencoded by the composition is sufficient to retard the growth ofabnormal cells, promote tumor progression reduction, prevent kidneyfailure, and increase survival in cancer-bearing animals. It is alsopossible to enhance the uptake of the composition (i.e. nucleic acidexpression construct) of this invention by placing a plurality ofelectrodes in a selected tissue, then delivering nucleic acid expressionconstruct to the selected tissue in an area that interposes theplurality of electrodes, and then applying a cell-transfecting pulse(e.g. electrical) to the selected tissue in an area of the selectedtissue where the nucleic acid expression construct was delivered.However, the cell-transfecting pulse need not be an electrical pulse, avascular pressure pulse can also be utilized. Electroporation, directinjection, gene gun, or gold particle bombardment are also used inspecific embodiments to deliver the composition that encodes the GHRH orbiological equivalent into the subject. The subject in this inventioncomprises a mammal (e.g. a human, a pig, a horse, a cow, a mouse, a rat,a monkey, a sheep, a goat, a dog, or a cat).

Additionally, the invention relates to a plasmid-mediatedsupplementation method for the treatment of anemia, wasting, tumorgrowth, immune dysfunction, kidney failure and/or life extension for thechronically ill subject. Anemia refers to a condition in which there isa reduction of the number, volume, or both of red blood corpuscles or ofthe total amount of hemoglobin in the bloodstream, resulting inpaleness, generalized weakness, etc. of the subject. Wasting of asubject can be defined as decreased body weight that is characterized bysignificant loss of both adipose tissue and muscle mass, which makesweight gain especially difficult for patients with a progressive disease(e.g. cancer, AIDS, etc.). Anemia, wasting, tumor growth, immunedysfunction, kidney failure and decreased life expectancy can be relatedto a specific disease or the effects of a disease treatment. Morespecifically, this invention pertains to a method for delivering aheterologous nucleic acid sequence encoding growth hormone releasinghormone (“GHRH”) or biological equivalent thereof into the cells of thesubject (e.g. somatic, stem, or germ cells) and allowing expression ofthe encoded GHRH or biological equivalent gene to occur while themodified cells are within the subject. The subsequent expression of theGHRH or biological equivalent thereof is regulated by a tissue specificpromoter (e.g. muscle), and/or by a regulator protein that contains amodified ligand-binding domain (e.g. molecular switch), which will onlybe active when the correct modified ligand (e.g. mifepistone) isexternally administered into the subject. The extracranial expressionand ensuing release of GHRH or biological equivalent thereof by themodified cells can be used to treat anemia, wasting, tumor growth,immune dysfunction, kidney failure and life extension for thechronically ill subject. The preferred means to deliver the GHRH orbiological equivalent thereof is by electroporation.

Recombinant GH replacement therapy is widely used clinically, withbeneficial effects, but generally, the doses are supraphysiological.Such elevated doses of recombinant GH are associated with deleteriousside-effects, for example, up to 30% of the recombinant GH treatedpatients report a higher frequency of insulin resistance (Blethen, 1995;Verhelst et al., 1997) or accelerated bone epiphysis growth and closurein pediatric patients (Blethen and Rundle, 1996). In addition, molecularheterogeneity of circulating GH may have important implications ingrowth and homeostasis, which can lead to a less potent GH that has areduced ability to stimulate the prolactin receptor (Satozawa et al.,2000; Tsunekawa et al., 1999; Wada et al., 1998). These unwanted sideeffects result from the fact that treatment with recombinant exogenousGH protein raises basal levels of GH and abolishes the natural episodicpulses of GH. In contradistinction, no side effects have been reportedfor recombinant GHRH therapies. The normal levels of GHRH in thepituitary portal circulation range from about 150-to-800 pg/ml, whilesystemic circulating values of the hormone are up to about 100-500pg/ml. Some patients with acromegaly caused by extracranial tumors havelevel that is nearly 10 times as high (e.g. 50 ng/ml of immunoreactiveGHRH) (Thomer et al., 1984). Long-term studies using recombinant GHRHtherapies (1-5 years) in children and elderly humans have shown anabsence of the classical GH side-effects, such as changes in fastingglucose concentration or, in pediatric patients, the accelerated boneepiphysal growth and closure or slipping of the capital femoralepiphysis (Chevalier et al., 2000) (Duck et al., 1992; Vittone et al.,1997). Numerous studies in humans, sheep or pigs showed that continuousinfusion with recombinant GHRH protein restores the normal GH patternwithout desensitizing GHRH receptors or depleting GH supplies (Dubreuilet al., 1990). As this system is capable of a degree of feed-back whichis abolished in the GH therapies, GHRH recombinant protein therapy maybe more physiological than GH therapy. However, due to the shorthalf-life of GHRH in vivo, frequent (one to three times per day)intravenous, subcutaneous or intranasal (requiring 300-fold higher dose)administrations are necessary (Evans et al., 1985; Thorner et al.,1986). Thus, as a chronic therapy, recombinant GHRH proteinadministration is not practical. A gene transfer approach, however couldovercome this limitations to GHRH use. Moreover, a wide range of dosescan be therapeutic. The choice of GHRH for a gene therapeuticapplication is favored by the fact that the gene, cDNA and native andseveral mutated molecules have been characterized for the pig and otherspecies (Bohlen et al., 1983; Guillemin et al., 1982), and themeasurement of therapeutic efficacy is straightforward and unequivocal.

Among the non-viral techniques for gene transfer in vivo, the directinjection of plasmid DNA into muscle is simple, inexpensive, and safe.The inefficient DNA uptake into muscle fibers after simple directinjection hag led to relatively low expression levels (Prentice et al.,1994; Wells et al., 1997) In addition, the duration of the transgeneexpression has been short (Wolff et al., 1990). The most successfulprevious clinical applications have been confined to vaccines (Danko andWolff, 1994; Tsurumi et al., 1996). Recently, significant progress toenhance plasmid delivery in vivo and subsequently to achievephysiological levels of a secreted protein was obtained using theelectroporation technique. Recently, significant progress has beenobtained using electroporation to enhance plasmid delivery in vivo.Electroporation has been used very successfully to transfect tumor cellsafter injection of plasmid (Lucas et al., 2002; Matsubara et al., 2001)or to deliver the anti-tumor drug bleomycin to cutaneous andsubcutaneous tumors in humans (Gehl et al., 1998; Heller et al., 1996).Electroporation also has been extensively used in mice (Lesbordes etal., 2002; Lucas et al., 2001; Vilquin et al., 2001), rats (Terada etal., 2001; Yasui et al., 2001), and dogs (Fewell et al., 2001) todeliver therapeutic genes that encode for a variety of hormones,cytokines or enzymes. Our previous studies using growth hormonereleasing hormone (GHRH) showed that plasmid therapy withelectroporation is scalable and represents a promising approach toinduce production and regulated secretion of proteins in large animalsand humans (Draghia-Akli et al., 1999; Draghia-Akli et al., 2002).Electroporation also has been extensively used in rodents and othersmall animals (Bettan et al., 2000; Yin and Tang, 2001). It has beenobserved that the electrode configuration affects the electric fielddistribution, and subsequent results (Gehl et al., 1999; Miklavcic etal., 1998). Preliminary experiments indicated that for a large animalmodel, needle electrodes give consistently better reproducible resultsthan external caliper electrodes.

The ability of electroporation to enhance plasmid uptake into theskeletal muscle has been well documented, as described above. Inaddition, plasmid formulated with PLG or polyvinylpyrrolidone (“PVP”)has been observed to increase gene transfection and consequently geneexpression to up to 10 fold in the skeletal muscle of mice, rats anddogs (Fewell et al., 2001; Mumper et al., 1998). Although not wanting tobe bound by theory, PLG will increase the transfection of the plasmidduring the electroporation process, not only by stabilizing the plasmidDNA, and facilitating the intracellular transport through the membranepores, but also through an active mechanism. For example, positivelycharged surface proteins on the cells could complex the negativelycharged PLG linked to plasmid DNA through protein-protein interactions.When an electric field is applied, the surface proteins reversedirection and actively internalize the DNA molecules, process thatsubstantially increases the transfection efficiency.

The plasmid supplementation approach to treat anemia, wasting, tumorgrowth, immune dysfunction, kidney failure and life extension for thechronically ill subject that is described herein offers advantages overthe limitations of directly injecting recombinant GH or GHRH protein.Expression of novel biological equivalents of GHRH that are serumprotease resistant can be directed by an expression plasmid controlledby a synthetic muscle-specific promoter. Expression of such GHRH orbiological equivalent thereof elicited high GH and IGF-I levels insubjects that have had the encoding sequences delivered into the cellsof the subject by intramuscular injection and in vivo electroporation.Although in vivo electroporation is the preferred method of introducingthe heterologous nucleic acid encoding system into the cells of thesubject, other methods exist and should be known by a person skilled inthe art (e.g. electroporation, lipofectamine, calcium phosphate, ex vivotransformation, direct injection, DEAE dextran, sonication loading,receptor mediated transfection, microprojectile bombardment, etc.). Forexample, it may also be possible to introduce the nucleic acid sequencethat encodes the GHRH or functional biological equivalent thereofdirectly into the cells of the subject by first removing the cells fromthe body of the subject or donor, maintaining the cells in culture, thenintroducing the nucleic acid encoding system by a variety of methods(e.g. electroporation, lipofectamine, calcium phosphate, ex vivotransformation, direct injection, DEAE dextran, sonication loading,receptor mediated transfection, microprojectile bombardment, etc.), andfinally reintroducing the modified cells into the original subject orother host subject (the ex vivo method). The GHRH sequence can be clonedinto an adenovirus vector or an adeno-associated vector and delivered bysimple intramuscular injection, or intravenously or intra-arterially.Plasmid DNA carrying the GHRH sequence can be complexed with cationiclipids or liposomes and delivered intramuscularly, intravenously orsubcutaneous.

Administration as used herein refers to the route of introduction of avector or carrier of DNA into the body. Administration can be directlyto a target tissue or by targeted delivery to the target tissue aftersystemic administration. In particular, the present invention can beused for treating disease by administration of the vector to the body inorder to establishing controlled expression of any specific nucleic acidsequence within tissues at certain levels that are useful for plasmidmediated supplementation. The preferred means for administration ofvector and use of formulations for delivery are described above.

Muscle cells have the unique ability to take up DNA from theextracellular space after simple injection of DNA particles as asolution, suspension, or colloid into the muscle. Expression of DNA bythis method can be sustained for several months. DNA uptake in musclecells is further enhance utilizing in vivo electroporation.

Delivery of formulated DNA vectors involves incorporating DNA intomacromolecular complexes that undergo endocytosis by the target cell.Such complexes may include lipids, proteins, carbohydrates, syntheticorganic compounds, or inorganic compounds. The characteristics of thecomplex formed with the vector (size, charge, surface characteristics,composition) determine the bioavailability of the vector within thebody. Other elements of the formulation function as ligands thatinteract with specific receptors on the surface or interior of the cell.Other elements of the formulation function to enhance entry into thecell, release from the endosome, and entry into the nucleus.

Delivery can also be through use of DNA transporters. DNA transportersrefer to molecules which bind to DNA vectors and are capable of beingtaken up by epidermal cells. DNA transporters contain a molecularcomplex capable of non-covalently binding to DNA and efficientlytransporting the DNA through the cell membrane. It is preferable thatthe transporter also transport the DNA through the nuclear membrane.See, e.g., the following applications all of which (including drawings)are hereby incorporated by reference herein: (1) Woo et al., U.S. Pat.No. 6,150,168 entitled: “A DNA Transporter System and Method of Use;”(2) Woo et al., PCT/US93/02725, entitled “A DNA Transporter System andmethod of Use”, filed Mar. 19, 1993; (3) Woo et al., U.S. Pat. No.6,177,554 “Nucleic Acid Transporter Systems and Methods of Use;” (4)Szoka et al., U.S. Pat. No. 5,955,365 entitled “Self-AssemblingPolynucleotide Delivery System;” and (5) Szoka et al., PCT/US93/03406,entitled “Self-Assembling Polynucleotide Delivery System”, filed Apr. 5,1993.

Another method of delivery involves a DNA transporter system. The DNAtransporter system consists of particles containing several elementsthat are independently and non-covalently bound to DNA. Each elementconsists of a ligand which recognizes specific receptors or otherfunctional groups such as a protein complexed with a cationic group thatbinds to DNA. Examples of cations which may be used are spermine,spermine derivatives, histone, cationic peptides and/or polylysine; oneelement is capable of binding both to the DNA vector and to a cellsurface receptor on the target cell. Examples of such elements areorganic compounds which interact with the asialoglycoprotein receptor,the folate receptor, the mannose-6-phosphate receptor, or the carnitinereceptor. A second element is capable of binding both to the DNA vectorand to a receptor on the nuclear membrane. The nuclear ligand is capableof recognizing and transporting a transporter system through a nuclearmembrane. An example of such ligand is the nuclear targeting sequencefrom SV40 large T antigen or histone. A third element is capable ofbinding to both the DNA vector and to elements which induce episomallysis. Examples include inactivated virus particles such as adenovirus,peptides related to influenza virus hemagglutinin, or the GALA peptidedescribed in the Skoka patent cited above.

Administration may also involve lipids. The lipids may form liposomeswhich are hollow spherical vesicles composed of lipids arranged inunilamellar, bilamellar, or multilamellar fashion and an internalaqueous space for entrapping water soluble compounds, such as DNA,ranging in size from 0.05 to several microns in diameter. Lipids may beuseful without forming liposomes. Specific examples include the use ofcationic lipids and complexes containing DOPE which interact with DNAand with the membrane of the target cell to facilitate entry of DNA intothe cell.

Gene delivery can also be performed by transplanting geneticallyengineered cells. For example, immature muscle cells called myoblastsmay be used to carry genes into the muscle fibers. Myoblast geneticallyengineered to express recombinant human growth hormone can secrete thegrowth hormone into the animal's blood. Secretion of the incorporatedgene can be sustained over periods up to 3 months.

Myoblasts eventually differentiate and fuse to existing muscle tissue.Because the cell is incorporated into an existing structure, it is notjust tolerated but nurtured. Myoblasts can easily be obtained by takingmuscle tissue from an individual who needs plasmid-mediatedsupplementation and the genetically engineered cells can also be easilyput back with out causing damage to the patient's muscle. Similarly,keratinocytes may be used to delivery genes to tissues. Large numbers ofkeratinocytes can be generated by cultivation of a small biopsy. Thecultures can be prepared as stratified sheets and when grafted tohumans, generate epidermis which continues to improve in histotypicquality over many years. The keratinocytes are genetically engineeredwhile in culture by transfecting the keratinocytes with the appropriatevector. Although keratinocytes are separated from the circulation by thebasement membrane dividing the epidermis from the dermis, humankeratinocytes secrete into circulation the protein produced.

Delivery may also involve the use of viral vectors. For example, anadenoviral vector may be constructed by replacing the E1 region of thevirus genome with the vector elements described in this inventionincluding promoter, 5′UTR, 3′UTR and nucleic acid cassette andintroducing this recombinant genome into 293 cells which will packagethis gene into an infectious virus particle. Virus from this cell maythen be used to infect tissue ex vivo or in vivo to introduce the vectorinto tissues leading to expression of the gene in the nucleic acidcassette.

Although not wanting to be bound by theory, it is believed that in orderto provide an acceptable safety margin for the use of such heterologousnucleic acid sequences in humans, a regulated gene expression system ismandated to possess low levels of basal expression of GHRH, and stillretain a high ability to induce. Thus, target gene expression can beregulated by incorporating molecular switch technology as schematicallydiagramed in FIG. 9 and further discussed in Example 1. The HV-GHRH orbiological equivalent molecule displays a high degree of stability inserum, with a half-life of 6 hours, versus the natural GHRH, that has a6-12 minutes half-life. Thus, by combining the powerful electroporationDNA delivery method with stable and regulable GHRH or biologicalequivalent encoded nucleic acid sequences, a therapy can be utilizedthat will reverse chronic wasting, allow the subject to gain weight, andextend the subject's life expectancy.

I. Vectors

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell wherein, in some embodiments, it can be replicated. A nucleicacid sequence can be native to the animal, or it can be “exogenous,”which means that it is foreign to the cell into which the vector isbeing introduced or that the sequence is homologous to a sequence in thecell but in a position within the host cell nucleic acid in which thesequence is ordinarily not found. Vectors include plasmids, cosmids,viruses (bacteriophage, animal viruses, and plant viruses), linear DNAfragments, and artificial chromosomes (e.g., YACs), although in apreferred embodiment the vector contains substantially no viralsequences. One of skill in the art would be well equipped to construct avector through standard recombinant techniques (see, for example,Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated hereinby reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operatively linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

II. Plasmid Vectors

In certain embodiments, a linear DNA fragment from a plasmid vector iscontemplated for use to transfect a eukaryotic cell, particularly amammalian cell. In general, plasmid vectors containing replicon andcontrol sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries a replication site, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. In anon-limiting example, E. coli is often transformed using derivatives ofpBR322, a plasmid derived from an E. coli species. pBR322 contains genesfor ampicillin and tetracycline resistance and thus provides easy meansfor identifying transformed cells. The pBR plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, forexample, promoters which can be used by the microbial organism forexpression of its own proteins. A skilled artisan recognizes that anyplasmid in the art may be modified for use in the methods of the presentinvention. In a specific embodiment, for example, a GHRH vector used forthe therapeutic applications is derived from pBlueScript KS+ and has akanamycin resistance gene.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEM™-11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as, for example,E. coli LE392.

Further useful plasmid vectors include pIN vectors (Inouye et al.,1985); and pGEX vectors, for use in generating glutathione S-transferase(“GST”) soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with β-galactosidase,ubiquitin, and the like.

Bacterial host cells, for example, E. coli, comprising the expressionvector, are grown in any of a number of suitable media, for example, LB.The expression of the recombinant protein in certain vectors may beinduced, as would be understood by those of skill in the art, bycontacting a host cell with an agent specific for certain promoters,e.g., by adding IPTG to the media or by switching incubation to a highertemperature. After culturing the bacteria for a further period,generally of between 2 and 24 h, the cells are collected bycentrifugation and washed to remove residual media.

III. Promoters and Enhancers

A promoter is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription of a gene productare controlled. It may contain genetic elements at which regulatoryproteins and molecules may bind, such as RNA polymerase and othertranscription factors, to initiate the specific transcription a nucleicacid sequence. The phrases “operatively positioned,” “operativelylinked,” “under control,” and “under transcriptional control” mean thata promoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30-110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant, synthetic or heterologous promoter, which refers to apromoter that is not normally associated with a nucleic acid sequence inits natural environment. A recombinant, synthetic or heterologousenhancer refers also to an enhancer not normally associated with anucleic acid sequence in its natural environment. Such promoters orenhancers may include promoters or enhancers of other genes, andpromoters or enhancers isolated from any other virus, or prokaryotic oreukaryotic cell, and promoters or enhancers not “naturally occurring,”i.e., containing different elements of different transcriptionalregulatory regions, and/or mutations that alter expression. For example,promoters that are most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase), lactose and tryptophan (trp)promoter systems. In addition to producing nucleic acid sequences ofpromoters and enhancers synthetically, sequences may be produced usingrecombinant cloning and/or nucleic acid amplification technology,including PCR™, in connection with the compositions disclosed herein(see U.S. Pat. Nos. 4,683,202 and 5,928,906, each incorporated herein byreference). Furthermore, it is contemplated the control sequences thatdirect transcription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

Additionally any promoter/enhancer combination (as per, for example, theEukaryotic Promoter Data Base EPDB, http://www.epd.isb-sib.ch/) couldalso be used to drive expression. Use of a T3, T7 or SP6 cytoplasmicexpression system is another possible embodiment. Eukaryotic cells cansupport cytoplasmic transcription from certain bacterial promoters ifthe appropriate bacterial polymerase is provided, either as part of thedelivery complex or as an additional genetic expression construct.

Tables 1 and 2 list non-limiting examples of elements/promoters that maybe employed, in the context of the present invention, to regulate theexpression of a RNA. Table 2 provides non-limiting examples of inducibleelements, which are regions of a nucleic acid sequence that can beactivated in response to a specific stimulus.

TABLE 1 Promoter and/or Enhancer Promoter/Enhancer Relevant ReferencesImmunoglobulin Heavy Chain Immunoglobulin Light Chain T-Cell ReceptorHLA DQ a and/or DQ β β-Interferon Interleukin-2 Interleukin-2 ReceptorMHC Class II 5 MHC Class II HLA-Dra β-Actin (Kawamoto et al., 1988;Kawamoto et al., 1989) Muscle Creatine Kinase (MCK) (Horlick andBenfield, 1989; Jaynes et al., 1988) Prealbumin (Transthyretin) ElastaseI Metallothionein (MTII) (Inouye et al., 1994; Narum et al., 2001;Skroch et al., 1993) Collagenase Albumin (Pinkert et al., 1987; Troncheet al., 1989) α-Fetoprotein γ-Globin β-Globin (Tronche et al., 1990;Trudel and Costantini, 1987) c-fos c-HA-ras Insulin (German et al.,1995; Ohlsson et al., 1991) Neural Cell Adhesion Molecule (NCAM)α₁-Antitrypsin H2B (TH2B) Histone Mouse and/or Type I CollagenGlucose-Regulated Proteins (GRP94 and GRP78) Rat Growth Hormone (Larsenet al., 1986) Human Serum Amyloid A (SAA) Troponin I (TN I) (Lin et al.,1991; Yutzey and Konieczny, 1992) Platelet-Derived Growth Factor (Pechet al., 1989) (PDGF) Duchenne Muscular Dystrophy (Klamut et al., 1990;Klamut et al., 1996) SV40 Polyoma Retroviruses Papilloma Virus HepatitisB Virus Human Immunodeficiency Virus Cytomegalovirus CMV (Boshart etal., 1985; Dorsch- Hasler et al., 1985) Gibbon Ape Leukemia VirusSynthetic muscle specific promoters (Draghia-Akli et al., 1999; (c5-12,c1-28 Draghia-Akli et al., 2002; Li etal., 1999)

TABLE 2 Element/Inducer Element Inducer MT II Phorbol Ester (TFA) Heavymetals MMTV (mouse mammary Glucocorticoids tumor virus) β-InterferonPoly (rI)x / Poly (rc) Adenovirus 5 E2 E1A Collagenase Phorbol Ester(TPA) Stromelysin Phorbol Ester (TPA) SV40 Phorbol Ester (TPA) Murine MXGene Interferon, Newcastle Disease Virus GRP78 Gene A23187α-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene H-2κb InterferonHSP70 E1A, SV40 Large T Antigen Proliferin Phorbol Ester-TPA TumorNecrosis Factor α PMA Thyroid Stimulating Hormone Thyroid Hormone α Gene

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Nonlimiting examples of such regions include the human LIMK2 gene(Nomoto et al., 1999), the somatostatin receptor 2 gene (Kraus et al.,1998), murine epididymal retinoic acid-binding gene (Lareyre et al.,1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen(Liu et al., 2000; Tsumaki et al., 1998), DIA dopamine receptor gene(Lee et al., 1997), insulin-like growth factor II (Dai et al., 2001; Wuet al., 1997), and human platelet endothelial cell adhesion molecule-1(Almendro et al., 1996).

In a preferred embodiment, a synthetic muscle promoter is utilized, suchas SPc5-12 (Li et al., 1999), which contains a proximal serum responseelement (“SRE”) from skeletal α-actin, multiple MEF-2 sites, MEF-1sites, and TEF-1 binding sites, and greatly exceeds the transcriptionalpotencies of natural myogenic promoters. The uniqueness of such asynthetic promoter is a significant improvement over, for instance,issued patents concerning a myogenic promoter and its use (e.g. U.S.Pat. No. 5,374,544) or systems for myogenic expression of a nucleic acidsequence (e.g. U.S. Pat. No. 5,298,422). In a preferred embodiment, thepromoter utilized in the invention does not get shut off or reduced inactivity significantly by endogenous cellular machinery or factors.Other elements, including trans-acting factor binding sites andenhancers may be used in accordance with this embodiment of theinvention. In an alternative embodiment, a natural myogenic promoter isutilized, and a skilled artisan is aware how to obtain such promotersequences from databases including the National Center for BiotechnologyInformation (“NCBI”) GenBank database or the NCBI PubMed site. A skilledartisan is aware that these databases may be utilized to obtainsequences or relevant literature related to the present invention.

IV. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (“IRES”) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

V. Multiple Cloning Sites

Vectors can include a MCS, which is a nucleic acid region that containsmultiple restriction enzyme sites, any of which can be used inconjunction with standard recombinant technology to digest the vector(see, for example, (Carbonelli et al., 1999; Cocea, 1997; Levenson etal., 1998) incorporated herein by reference.) “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is widely understood by those of skill in the art.Frequently, a vector is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the vector. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology.

VI. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, (Chandler et al., 1997), herein incorporated by reference.)

VII. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (“polyA”)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

VIII. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal, skeletal alpha actin 3′UTR or the human orbovine growth hormone polyadenylation signal, convenient and known tofunction well in various target cells. Polyadenylation may increase thestability of the transcript or may facilitate cytoplasmic transport.

IX. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (“ARS”) can beemployed if the host cell is yeast.

X. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (“tk”) orchloramphenicol acetyltransferase (“CAT”) may be utilized. One of skillin the art would also know how to employ immunologic markers, possiblyin conjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

XI. Electroporation

In certain embodiments of the present invention, a nucleic acid isintroduced into an organelle, a cell, a tissue or an organism viaelectroporation. Electroporation involves the exposure of a suspensionof cells and DNA to a high-voltage electric discharge. In some variantsof this method, certain cell wall-degrading enzymes, such aspectin-degrading enzymes, are employed to render the target recipientcells more susceptible to transformation by electroporation thanuntreated cells (U.S. Pat. No. 5,384,253, incorporated herein byreference). Alternatively, recipient cells can be made more susceptibleto transformation by mechanical wounding and other methods known in theart.

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre-B lymphocytes have been transfected with humankappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocyteshave been transfected with the chloramphenicol acetyltransferase gene(Tur-Kaspa et al., 1986) in this manner.

The underlying phenomenon of electroporation is believed to be the samein all cases, but the exact mechanism responsible for the observedeffects has not been elucidated. Although not wanting to be bound bytheory, the overt manifestation of the electroporative effect is thatcell membranes become transiently permeable to large molecules, afterthe cells have been exposed to electric pulses. There are conduitsthrough cell walls, which under normal circumstances, maintain a restingtransmembrane potential of ca. 90 mV by allowing bi-directional ionicmigration.

Although not wanting to be bound by theory, electroporation makes use ofthe same structures, by forcing a high ionic flux through thesestructures and opening or enlarging the conduits. In prior art, metallicelectrodes are placed in contact with tissues and predeterminedvoltages, proportional to the distance between the electrodes areimposed on them. The protocols used for electroporation are defined interms of the resulting field intensities, according to the formulaE=V/d, where (“E”) is the field, (“V”) is the imposed voltage and (“d”)is the distance between the electrodes.

The electric field intensity E has been a very important value in priorart when formulating electroporation protocols for the delivery of adrug or macromolecule into the cell of the subject. Accordingly, it ispossible to calculate any electric field intensity for a variety ofprotocols by applying a pulse of predetermined voltage that isproportional to the distance between electrodes. However, a caveat isthat an electric field can be generated in a tissue with insulatedelectrodes (i.e. flow of ions is not necessary to create an electricfield). Although not wanting to be bound by theory, it is the currentthat is necessary for successful electroporation not electric field perse.

During electroporation, the heat produced is the product of theinterelectrode impedance, the square of the current, and the pulseduration. Heat is produced during electroporation in tissues and can bederived as the product of the inter-electrode current, voltage and pulseduration. The protocols currently described for electroporation aredefined in terms of the resulting field intensities E, which aredependent on short voltage pulses of unknown current. Accordingly, theresistance or heat generated in a tissue cannot be determined, whichleads to varied success with different pulsed voltage electroporationprotocols with predetermined voltages. The ability to limit heating ofcells across electrodes can increase the effectiveness of any givenelectroporation voltage pulsing protocol. For example, prior art teachesthe utilization of an array of six needle electrodes utilizing apredetermined voltage pulse across opposing electrode pairs. Thissituation sets up a centralized pattern during an electroporation eventin an area where congruent and intersecting overlap points develop.Excessive heating of cells and tissue along electroporation path willkill the cells, and limit the effectiveness of the protocol. However,symmetrically arranged needle electrodes without opposing pairs canproduce a decentralized pattern during an electroporation event in anarea where no congruent electroporation overlap points can develop.

Controlling the current flow between electrodes allows one to determinethe relative heating of cells. Thus, it is the current that determinesthe subsequent effectiveness of any given pulsing protocol, and not thevoltage across the electrodes. Predetermined voltages do not producepredetermined currents, and prior art does not provide a means todetermine the exact dosage of current, which limits the usefulness ofthe technique. Thus, controlling an maintaining the current in thetissue between two electrodes under a threshold will allow one to varythe pulse conditions, reduce cell heating, create less cell death, andincorporate macromolecules into cells more efficiently when compared topredetermined voltage pulses.

One embodiment of the present invention to overcome the above problem byproviding a means to effectively control the dosage of electricitydelivered to the cells in the inter-electrode space by preciselycontrolling the ionic flux that impinges on the conduits in the cellmembranes. The precise dosage of electricity to tissues can becalculated as the product of the current level, the pulse length and thenumber of pulses delivered. Thus, a specific embodiment of the presentinvention can deliver the electroporative current to a volume of tissuealong a plurality of paths without, causing excessive concentration ofcumulative current in any one location, thereby avoiding cell deathowing to overheating of the tissue.

Although not wanting to be bound by theory, the nature of the voltagepulse to be generated is determine by the nature of tissue, the size ofthe selected tissue and distance between electrodes. It is desirablethat the voltage pulse be as homogenous as possible and of the correctamplitude. Excessive field strength results in the lysing of cells,whereas a low field strength results in reduced efficacy ofelectroporation. Some electroporation devices utilize the distancebetween electrodes to calculate the electric field strength andpredetermined voltage pulses for electroporation. This reliance onknowing the distance between electrodes is a limitation to the design ofelectrodes. Because the programmable current pulse controller willdetermine the impedance in a volume of tissue between two electrodes,the distance between electrodes is not a critical factor for determiningthe appropriate electrical current pulse. Therefore, an alternativeembodiment of a needle electrode array design would be one that isnon-symmetrical. In addition, one skilled in the art can imagine anynumber of suitable symmetrical and non-symmetrical needle electrodearrays that do not deviate from the spirit and scope of the invention.The depth of each individual electrode within an array and in thedesired tissue could be varied with comparable results. In addition,multiple injection sites for the macromolecules could be added to theneedle electrode array.

XII. Restriction Enzymes

In some embodiments of the present invention, a linear DNA fragment isgenerated by restriction enzyme digestion of a parent DNA molecule.Examples of restriction enzymes are provided in the following table.

Name Recognition Sequence AatII GACGTC Acc65 I GGTACC Acc I GTMKAC Aci ICCGC Acl I AACGTT Afe I AGCGCT Afl II CTTAAG Afl III ACRYGT Age I ACCGGTAhd I GACNNNNNGTC Alu I AGCT Alw I GGATC AlwN I CAGNNNCTG Apa I GGGCCCApaL I GTGCAC Apo I RAATTY Asc I GGCGCGCC Ase I ATTAAT Ava I CYCGRG AvaII GGWCC Avr II CCTAGG Bae I NACNNNNGTAPyCN Bam HI GGATCC Ban I GGYRCCBan II GRGCYC Bbs I GAAGAC Bbv I GCAGC BbvC I CCTCAGC Bcg I CGANNNNNNTGCBciV I GTATCC Bcl I TGATCA Bfa I CTAG Bgl I GCCNNNNNGGC Bgl II AGATCTBlp I GCTNAGC Bmr I ACTGGG Bpm I CTGGAG BsaA I YACGTR BsaB I GATNNNNATCBsaH I GRCGYC Bsa I GGTCTC BsaJ I CCNNGG BsaW I WCCGGW BseR I GAGGAG BsgI GTGCAG BsiE I CGRYCG BsiHKA I GWGCWC BsiW I CGTACG Bsl I CCNNNNNNNGGBsmA I GTCTC BsmB I CGTCTC BsmF I GGGAC Bsm I GAATGC BsoB I CYCGRG Bsp1286 I GDGCHC BspD I ATCGAT BspE I TCCGGA BspH I TCATGA BspM I ACCTGCBsrB I CCGCTC BsrD I GCAATG BsrF I RCCGGY BsrG I TGTACA Bsr I ACTGG BssHII GCGCGC BssK I CCNGG Bst4C I ACNGT BssS I CACGAG BstAP I GCANNNNNTGCBstB I TTCGAA BstE II GGTNACC BstF5 I GGATGNN BstN I CCWGG BstU I CGCGBstX I CCANNNNNNTGG BstY I RGATCY BstZ17 I GTATAC Bsu36 I CCTNAGG Btg ICCPuPyGG Btr I CACGTG Cac8 I GCNNGC Cla I ATCGAT Dde I CTNAG Dpn I GATCDpn II GATC Dra I TTTAAA Dra III CACNNNGTG Drd I GACNNNNNNGTC Eae IYGGCCR Eag I CGGCCG Ear I CTCTTC Eci I GGCGGA EcoN I CCTNNNNNAGG EcoO109I RGGNCCY EcoR I GAATTC EcoR V GATATC Fau I CCCGCNNNN Fnu4H I GCNGC FokI GGATG Fse I GGCCGGCC Fsp I TGCGCA Hae II RGCGCY Hae III GGCC Hga IGACGC Hha I GCGC Hinc II GTYRAC Hind III AAGCTT Hinf I GANTC HinP1 IGCGC Hpa I GTTAAC Hpa II CCGG Hph I GGTGA Kas I GGCGCC Kpn I GGTACC MboI GATC Mbo II GAAGA Mfe I CAATTG Mlu I ACGCGT Mly I GAGTCNNNNN Mnl ICCTC Msc I TGGCCA Mse I TTAA Msl I CAYNNNNRTG MspA1 I CMGCKG Msp I CCGGMwo I GCNNNNNNNGC Nae I GCCGGC Nar I GGCGCC Nci I CCSGG Nco I CCATGG NdeI CATATG NgoMI V GCCGGC Nhe I GCTAGC Nla III CATG Nla IV GGNNCC Not IGCGGCCGC Nru I TCGCGA Nsi I ATGCAT Nsp I RCATGY Pac I TTAATTAA PaeR7 ICTCGAG Pci I ACATGT PflF I GACNNNGTC PflM I CCANNNNNTGG PleI GAGTC Pme IGTTTAAAC Pml I CACGTG PpuM I RGGWCCY PshA I GACNNNNGTC Psi I TTATAA PspGI CCWGG PspOM I GGGCCC Pst I CTGCAG Pvu I CGATCG Pvu II CAGCTG Rsa IGTAC Rsr II CGGWCCG Sac I GAGCTC Sac II CCGCGG Sal I GTCGAC Sap IGCTCTTC Sau3A I GATC Sau96 I GGNCC Sbf I CCTGCAGG Sca I AGTACT ScrF ICCNGG SexA I ACCWGGT SfaN I GCATC Sfc I CTRYAG Sfi I GGCCNNNNNGGCC Sfo IGGCGCC SgrA I CRCCGGYG Sma I CCCGGG Sml I CTYRAG SnaB I TACGTA Spe IACTAGT Sph I GCATGC Ssp I AATATT Stu I AGGCCT Sty I CCWWGG Swa IATTTAAAT Taq I TCGA Tfi I GAWTC Tli I CTCGAG Tse I GCWGC Tsp45 I GTSACTsp509 I AATT TspR I CAGTG Tth111 I GACNNNGTC Xba I TCTAGA Xcm ICCANNNNNNNNNTG G Xho I CTCGAG Xma I CCCGGG Xmn I GAANNNNTTC

The term “restriction enzyme digestion” of DNA as used herein refers tocatalytic cleavage of the DNA with an enzyme that acts only at certainlocations in the DNA. Such enzymes are called restriction endonucleases,and the sites for which each is specific is called a restriction site.The various restriction enzymes used herein are commercially availableand their reaction conditions, cofactors, and other requirements asestablished by the enzyme suppliers are used. Restriction enzymescommonly are designated by abbreviations composed of a capital letterfollowed by other letters representing the microorganism from which eachrestriction enzyme originally was obtained and then a number designatingthe particular enzyme. In general, about 1 μg of plasmid or DNA fragmentis used with about 1-2 units of enzyme in about 20 μl of buffersolution. Appropriate buffers and substrate amounts for particularrestriction enzymes are specified by the manufacturer. Incubation ofabout 1 hour at 37° C. is ordinarily used, but may vary in accordancewith the supplier's instructions. After incubation, protein orpolypeptide is removed by extraction with phenol and chloroform, and thedigested nucleic acid is recovered from the aqueous fraction byprecipitation with ethanol. Digestion with a restriction enzyme may befollowed with bacterial alkaline phosphatase hydrolysis of the terminal5′ phosphates to prevent the two restriction cleaved ends of a DNAfragment from “circularizing” or forming a closed loop that would impedeinsertion of another DNA fragment at the restriction site. Unlessotherwise stated, digestion of plasmids is not followed by 5′ terminaldephosphorylation. Procedures and reagents for dephosphorylation areconventional as described in the art.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Construction of DNA Vectors and Methods in Animal Subject

In order to treat anemia; increase total red blood cell mass; reversethe wasting; reverse abnormal weight loss; treat immune dysfunction;reverse the suppression of lymphopoesis; or extend life expectancy forthe chronically ill subject, it was first necessary to design severalGHRH expression constructs. Briefly, the plasmid vectors contained themuscle specific synthetic promoter SPc5-12 (Li et al., 1999) attached toa wild type or analog porcine GHRH. The analog GHRH sequences weregenerated by site directed mutagenesis as described in methods section.Nucleic acid sequences encoding GHRH or analog were cloned into theBamHI/HindIII sites of pSPc5-12 plasmid, to generate pSP-GHRH. Otherelements contained in the plasmids include a 3′ untranslated region ofgrowth hormone and an SV40 3′UTR from pSEAP-2 Basic Vector as describedin the methods section. The unique nucleic acid sequences for theconstructs used are shown in FIG. 1.

DNA Constructs: Plasmid vectors containing the muscle specific syntheticpromoter SPc5-12 (SEQ ID NO: 7) were previously described (Li et al.,1999). Wild type and mutated porcine GHRH cDNAs were generated by sitedirected mutagenesis of GHRH cDNA (SEQ ID NO: 9) (Altered Sites II invitro Mutagenesis System, Promega, Madison, Wis.), and cloned into theBamHI/Hind III sites of pSPc5-12, to generate pSP-wt-GHRH (SEQ ID NO:15), or pSP-HV-GHRH (SEQ ID NO: 11), respectively. The 3″ untranslatedregion (3″ UTR) of growth hormone was cloned downstream of GHRH cDNA.The resultant plasmids contained mutated coding region for GHRH, and theresultant amino acid sequences were not naturally present in mammals.Although not wanting to be bound by theory, the effects on treatinganemia; increasing total red blood cell mass in a subject; reversing thewasting; reversing abnormal weight loss; decreasing tumor growth;preventing kidney failure; treating immune dysfunction; reversing thesuppression of lymphopoesis; or extending life expectancy for thechronically ill subject are determined ultimately by the circulatinglevels of mutated hormones. Several different plasmids that encodeddifferent mutated amino acid sequences of GHPd-t or functionalbiological equivalent thereof are as follows:

Plasmid Encoded Amino Acid Sequence wt-GHRHYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGERNQEQGA-OH (SEQID#10) HV-GHRHHVDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-OH (SEQID#1) TI-GHRHYIDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-OH (SEQID#2) TV-GHRHYVDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-OH (SEQID#3) 15/27/28-GHRHYADAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-OH (SEQID#4)

In general, the encoded GHRH or functional biological equivalent thereofis of formula:-X₁-X₂-DAIFTNSYRKVL-X₃-QLSARKLLQDI-X₄-X₅-RQQGERNQEQGA-OH(SEQ ID NO: 6) wherein: X₁ is a D- or L-isomer of an amino acid selectedfrom the group consisting of tyrosine (“Y”), or histidine (“H”); X₂ is aD- or L-isomer of an amino acid selected from the group consisting ofalanine (“A”), valine (“V”), or isoleucine (‘T’); X₃ is a D- or L-isomerof an amino acid selected from the group consisting of alanine (“A”) orglycine (“G”); X₄ is a D- or L-isomer of an amino acid selected from thegroup consisting of methionine (“M”), or leucine (“L”); X₅ is a D- orL-isomer of an amino acid selected from the group consisting of serime(“S”) or asparagine (“N”).

Another plasmid that was utilized included the pSP-SEAP construct (SEQID No: 16) that contains the Sadl/Hindill SPc5-12 fragment, SEAP geneand SV40 3″UTR from pSEAP-2 Basic Vector (Clontech Laboratories, Inc.;Palo Alto, Calif.).

The plasmids described above do not contain polylinker, IGF-I gene, askeletal alpha-actin promoter or a skeletal alpha actin 3′ UTR /NCR.Furthermore, these plasmids were introduced by muscle injection,followed by in vivo electroporation, as described below.

In terms of “functional biological equivalents”, it is well understoodby the skilled artisan that, inherent in the definition of a“biologically functional equivalent” protein and/or polynucleotide, isthe concept that there is a limit to the number of changes that may bemade within a defined portion of the molecule while retaining a moleculewith an acceptable level of equivalent biological activity. Functionalbiological equivalents are thus defined herein as those proteins (andpolynucleotides) in selected amino acids (or codons) may be substituted.A peptide comprising a functional biological equivalent of GHRH is apolypeptide that has been engineered to contain distinct amino acidsequences while simultaneously having similar or improved biologicallyactivity when compared to GHRH. For example one biological activity ofGHRH is to facilitate growth hormone (“GH”) secretion in the subject.

Large Animal Studies: Healthy Dogs: A group of 4 dogs (2 males and 2females) were used as controls and 3 groups of 8 dogs (4 males and 4females) were injected with the pSP-HV-GHRH system. The dogs wereinjected with vehicle alone (control), or 200 mcg, or 600 mcg or 1000mcg of pSP-HV-GHRH followed by caliper electroporation.

Cancer Dogs: Fifteen dogs with spontaneous malignancies were used inGHRH studies. The dogs were injected with 100 mcg/kg to a total of nomore than 1000 mcg pSP-HV-GHRH. Four dogs died or were euthanized atowner's request within the first three days after the plasmid injectionfrom unrelated complications of their advanced disease. The condition ofinclusion in our study was a survival of at least 14 days post-injection(in order to allow for plasmid activation and expression of GHRH fromthe skeletal muscle), when a second blood draw could be made. Elevendogs were analyzed in this study. The animals were under specifictreatment using either chemotherapy, radiotherapy or combination therapy(see FIG. 18). The animals were weighed and bled before the treatmentand at 9-27 and 28-56 days post-injection. At each time point completeCBC and metabolic profile was assessed by the same independentlaboratory (Antech Diagnostics, Irvine, Calif.). Wellness forms werecompleted by owners at each visit. Nineteen non-injected dogs withspontaneous malignancies, in treatment in the clinic in the same timewindow, were used as contemporary controls. The quality of life in thetreated patients increased. No adverse effects linked to the therapywere noted by owners. Three owners noticed a dramatic improvement in thegeneral well-being of the treated dog compared to pre-injection status.

Electroporation devices: A BTX T820 generator (BTX, division ofGenetronics Inc., CA) was used to deliver square wave pulses in allexperiments. We used voltage conditions of 100V/cm, 6 pulses, 60milliseconds per pulse. Two-needle electrodes (BTX, a division ofGenetronics Inc., CA) were used to deliver in vivo electric pulses. Inall injections the needles were completely inserted into the muscle.

Intramuscular injection of plasmid DNA in Canine subjects: Four groupsof healthy Canines (“dogs”) subjects, 8-12 kg in weight, were used forbiodistribution -toxicology studies. Three groups of 8 dogs (4 males and4 females) were injected with 200 mcg, 600 mcg and 1000 mcg ofpSP-HV-GHRH, respectively. A group of 4 dogs (2 males and 2 females)were used as controls. Animals were continuously monitored for sideeffects. In addition, two groups of dogs with cancer (spontaneousmalignancies) were used. Animals were maintained in accordance with NIHGuide, USDA and Animal Welfare Act guidelines, and approved by theBaylor College of Medicine IACUC.

Endotoxin-free plasmid (Qiagen Inc., Chatsworth, Calif., USA)preparation of pSPc5-12-HV-GHRH were diluted in PBS pH=7.4 or water to 5mg/mL. Dogs were injected before their regular treatment administration.For dogs on chemotherapy, the injection was administered at no less than5 days before/after the medication. The dogs were anesthetized withPropafol (Abbott Laboratories, IL) 4-8 mg/kg. While anesthetized, 100μg/kg to a maximum of 1 mg of plasmid was injected directly into thesemitendinosus muscle of dogs, using an 3/10 cc insulin syringe and29G1/2” needle (Becton-Dickinson, Franklin Lacks, N.J.). Two minutesafter injection, the injected muscle was electroporated, 6 pulses,100V/cm, 60 milliseconds/pulse, using a BTX T820 electroporator andtwo-needle electrodes (BTX, a division of Genetronics Inc., CA), asdescribed (Miklavcic et al., 2000). In all injections the needles werecompletely inserted into the muscle. Animals were allowed to recoverbefore rejoining their owners.

Although in vivo electroporation is the preferred method for deliveringthe nucleic acid constructs into the cells of the subject, suitablemethods for nucleic acid delivery for transformation of an organelle, acell, a tissue or an organism for use with the current invention arebelieved to include virtually any method by which a nucleic acid (e.g.,DNA) can be introduced into an organelle, a cell, a tissue or anorganism, as described herein or as would be known to one of ordinaryskill in the art. Such methods include, but are not limited to, directdelivery of DNA such as by ex vivo transfection (Nabel et al., 1989;Wilson et al., 1989), by injection (U.S. Pat. Nos. 5,994,624, 5,981,274,5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and5,580,859, each incorporated herein by reference), includingmicroinjection (Harland and Weintraub, 1985) U.S. Pat. No. 5,789,215,incorporated herein by reference); by electroporation (U.S. Pat. No.5,384,253, incorporated herein by reference; (Potter et al., 1984;Tur-Kaspa et al., 1986); by calcium phosphate precipitation (Chen andOkayama, 1987; Graham and van der Eb, 1973; Rippe et al., 1990); byusing DEAE-dextran followed by polyethylene glycol (Gopal, 1985); bydirect sonic loading (Fechheimer et al., 1987); by liposome mediatedtransfection (Hafez et al., 2001; Hamm et al., 2002; Madry et al., 2001;Raghavachari and Fahl, 2002; Wiethoff et al., 2001) andreceptor-mediated transfection (Wu and Wu, 1988a; Wu and Wu, 1988b); bymicroprojectile bombardment (PCT Application Nos. WO 94/09699 and95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318,5,538,877 and 5,538,880, and each incorporated herein by reference); byagitation with silicon carbide fibers ((Johnson et al., 1992); U.S. Pat.Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993); U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), andany combination of such methods. Through the application of techniquessuch as these, organelle(s), cell(s), tissue(s) or organism(s) may bestably or transiently transformed.

Body weight data: Animals were weighted before the plasmid injection andat 14, 28 and 56 days post-injection using the same calibrated scale.

Protein Metabolism, Bone metabolism and Blood values: Blood and urinesamples were collected before plasmid injection, and at 14, 28 and 56days post-injection, and analyzed for biochemistry, metabolisms andhormone. At each time point complete CBC and metabolic profile wasassessed by the same independent laboratory (Antech Diagnostics, Irvine,Calif.).

Plasma IGF-I: IGF-I levels were measured by heterologous humanradioimmunometric assay (Diagnostic System Lab., Webster, Tex.). Thesensitivity of the assay was 0.8 ng/ml; intra-assay and inter-assayvariation was 3.4% and 4.5% respectively.

Statistics: Data are analyzed using STATISTICA analysis package(StatSoft, Inc. Tulsa, Okla.). Values shown in the Figures are themean±s.e.m. Specific P values were obtained by comparison using ANOVA. AP<0.05 was set as the level of statistical significance.

Example 2 Low Voltage Electroporation for DNA Uptake and Expression inan Animal Subject

Direct intra-muscular plasmid DNA injection followed by electroporationis a method for the local and controlled delivery of plasmid DNA intoskeletal muscle. It has the advantage that is uses low plasmidquantities (as low as 0.1 mg), rather than the high quantities typicallyused with passive delivery modalities. Although not wanting to be boundby theory, the mechanism of the increased plasmid uptake byelectroporation probably occurs through newly created membrane poreswith or without protein active transport. Although not wanting to bebound by theory, the degree of permeabilization of the muscle cells isdependent on the electric field intensity, length of pulses, shape andtype of electrodes (Bureau et al., 2000) (Gilbert et al., 1997), andcell size (Somiari et al., 2000). Classical electrode configuration,plates or a pair of wire electrodes placed 4 mm apart were shown to beeffective in rodents, but in large mammals as pigs or humans theincreased resistance of the skin, the thickness of the subcutaneous fattissue, and the concern for tissue damage if the intensity of theelectric field would be proportionally increased, make these types ofelectrodes unpractical. The porcine or dog muscle fibers are quite largeand consequently more suitable for electropermeabilization than rodentmuscle. In this report, we show that a single injection various dosagesof GHRH or analog nucleic acid sequences followed by electroporationwith intramuscular applicators, in a large mammal is sufficient toproduce therapeutic plasma hormone levels, with biologically significanteffects that can treat anemia, reverse wasting, allow the subject togain weight, and extend life expectancy of the chronically ill.

The pSP-HV-GHRH system was delivered to the left tibialis anteriormuscle of healthy dogs via in vivo electroporation. A group of 4 dogs (2males and 2 females) were used as controls and 3 groups of 8 dogs (4males and 4 females) were injected with the pSP-HV-GHRH system. The dogswere injected with vehicle alone (control), or 200 mcg, or 600 mcg or1000 mcg of pSP-HV-GHRH followed by needle electroporation. Anindication of increased systemic levels of GHRH and GH is an increase inserum IGF-I concentration. Therefore, following 28 days post injectionblood serum was collected from the dogs were injected with vehicle alone(control), or 200 mcg, or 600 mcg or 1000 mcg of pSP-HV-GHRH and IGF-Ilevels were determined. The IGF-I levels for dogs injected with 600 mcgwere 3-fold higher than the control (vehicle alone) treated animals(FIG. 2). The increase in IGF-I levels was statistically significant(p<0.046). Although animals injected with 200 mcg and 1000 mcg ofplasmid showed higher IGF-I levels than controls, the IGF-I levels werelower than animals injected with 600 mcg. Increased IGF-I levelscorresponding to higher GHRH levels are in agreement with other studiesthat utilized recombinant porcine GH (“pGH”) in dogs. For example, therewere dose-related increased serum IGF-I levels (approximately 2-10-fold)that correlated with the elevated serum GH levels in pGH-treated dogs.

Although not wanting to be bound by theory, growth hormone releasinghormone (GHRH) stimulates the production and release from the anteriorpituitary of growth hormone (GH), which in turn stimulates theproduction of IGF-I from the liver and other target organs. Thus, anindication of increased systemic levels of GHRH and GH is an increase inserum IGF-I concentration. The level of serum IGF-I in healthy dogsinjected with 200, 600 and 1000 mcg of pSP-HV-GHRH were all higher 28days post-injection when compared to the pre-injection values. Dogsinjected with 600 mcg pSP-HV-GHRH showed the highest statisticallysignificant increase (e.g. greater than 90%, p <0.046) in IGF-I levels,which indicates that 600 mcg may be the optimal concentration forhealthy dogs.

Example 3 Increased Survival in Animal Subjects with Cancer

Eleven injected dogs with cancer had survived for at least 56 days afterthe injection, and complete data was collected in all cases. The dogsenrolled in the study were in a relatively advanced stage of theirdisease (206 days since the beginning of the therapy). The averagesurvival post-injection is listed in FIG. 18. At the time thisapplication was prepared, 8 out of the eleven treated dogs were stillalive (average survival post injection 150.6 days). The 19 control dogswere in a less advanced stage of disease and had an average survival of56 days after the initial diagnosis. In contrast, after the enrollmentinto the present study the average survival post enrolment was 162.5days. Five control animals died during this period. The quality of lifein the treated patients increased. No adverse effects linked to thetherapy were noted by owners. Three owners noticed a dramaticimprovement in the general well-being of the treated dog compared topre-injection status.

Example 4 Increased Weight Gain in Healthy Animal Subjects

In order to show that increased levels of GHRH or biological equivalentthereof could alter metabolism in large healthy animals, body weight wasdetermined. As shown in FIG. 4, the treated dogs had increased weightgain compared with controls fifty-six days post-injection. Althoughanimals injected with 200 mcg and 1000 mcg of plasmid showed higher bodyweights than controls, the weights were lower than animals injected with600 mcg. These results are a good indicator that the metabolism of thedogs injected with pSP-HV-GHRH was altered in a dose dependent manner.In addition, the additional weight gain associated with increasedproduction of GHRH also indicates that the levels of GH were increased.This observation is in agreement with other studies that utilizedrecombinant porcine GH (pGH) in dogs. In one of these studies,recombinant pGH was administered for 14-weeks in dogs. Porcine GH causedincreased body weight gain in mid - and high-dose groups (2.8 kg and 4.7kg, respectively), compared to 0.4 kg and 0.8 kg in control and low-dosegroups, respectively.

Example 5 GHRH or Biological Equivalent Treatment Improves ProteinUtilization in Healthy Dogs

FIG. 15 shows the changes in values associated with protein metabolismin healthy dogs injected with different concentrations of thepSP-HV-GHRH plasmid. Many values that indicate protein metabolism weremonitored for 56 days including: AST, ALT, T. bilirubin, Alk Phos, GGT,total protein, albumin, globulin, A/G ratio, Cholesterol, BUN andcreatinine. Groups of 8 dogs (4 males and 4 females) were injected with200 mcg, 600 mcg and 1000 mcg of pSP-HV-GHRH. A group of 4 dogs (2 malesand 2 females) were used as controls. Dogs injected with 200, and 600mcg of plasmid had increased total protein levels. All injected groupshave slightly decreased urea compared with controls, which is a sign ofimproved protein utilization.

Example 6 GHRH or Biological Equivalent Treatment Does Not AffectGlucose Metabolism in Healthy Animal Subjects.

FIG. 16 shows the changes in values associated with blood components inhealthy dogs injected with different concentrations of the pSP-HV-GHRHplasmid. Abbreviations are as follows: WBC—White Blood Cell; RBC—Redblood cell; HGB—hemoglobin; PCV—hematocrit, or packed cell volume;MCV—mean corpuscular volume; MCH—mean corpuscular hemoglobin; MCHC—meancorpuscular hemoglobin concentration; n %-% of neutrophils; lym %-% oflymphocytes; mono %-% of monocytes; eos %-% of eosinophils; Bas %-% ofbasophils; LDH—lactate dehydrogenase ; Prothom—prothrombine;Qnt—quantitative; Plat—platelets; BUN—blood urea nitrogen/urea.

Many blood component values were monitored for 56 days including: WBC,RBC, HGB, PCV, MCB, MCH, MCHC, n %, lym % mono % eos %, Bas %, LDH,Prothom, Qnt. Plat. Groups of 8 dogs (4 males and 4 females) wereinjected with 200 mcg, 600 mcg and 1000 mcg of pSP-HV-GHRH. A group of 4dogs (2 males and 2 females) were used as controls. No statisticaldifferences were found between experimental and control groups. However,circulation lymphocytes decreased with the increase in the plasmiddosage, sign of lymphocyte sequestration in the lymphatic organs.Importantly, glucose levels in all experimental groups and controls arewithin the normal range, which indicates that our therapy does notimpair glucose metabolism.

Example 7 GHRH or Biological Equivalent Treatment Effects BoneRemodeling

FIG. 17 shows the changes in values associated with bone metabolism inhealthy dogs injected with different concentrations of the pSP-HV-GHRHplasmid. The phosphorus, calcium and calcium/phosphorous ratio wasmonitored for 56 days post injection. Groups of 8 dogs (4 males and 4females) were injected with 200 mcg, 600 mcg and 1000 mcg ofpSP-HV-GHRH. A group of 4 dogs (2 males and 2 females) were used ascontrols. Dogs injected plasmid had an increased Ca/PO₄ ratio that wasproportional with the dosage of the treatment, which is a sign of boneremodeling.

Example 8 GHRH or Biological Equivalent Treatment Extends LifeExpectancy in Chronically Ill Subjects

FIG. 18 shows the diagnosis, specific therapy chart and survival fordogs with spontaneous cancer that were injected with differentconcentrations of the pSP-HV-GHRH plasmid. The study, group, treatment,dose, # of dogs, cancer type, and days survived post-treatment areindicated. Groups of dogs with spontaneous cancer were injected with 100mcg/Kg body weight to a total of no more than 1000 mcg of pSP-HV-GHRH.In addition, dogs treated with pSP-HV-GHRH had an improved quality oflife.

Example 9 GHRH or Biological Equivalent Treatment Positively AffectsImmune Function in Cancer Subjects

FIG. 19 shows the changes in values associated with blood components indogs with spontaneous cancer injected with different concentrations ofthe pSP-HV-GHRH plasmid. Many blood component values were monitored postinjection including: WBC/HPF, RBC/HBF, HGB, % PCV, MCV, MCH, MCHC, n %,lym % mono % eos %, Bas %. Groups of dogs with spontaneous cancer wereinjected with 100-1000 mcg/Kg body weight of pSP-HV-GHRH. Overall dogstreated with the pSV-HV-GHRH plasmid therapy, had increased RBChemoglobin and hematocrit levels two weeks post-injection compared withun-injected controls. In addition, treated dogs have a significantdecrease in the levels of circulation white blood cells (usuallyassociated with increase of white blood cells (WBC) in the lymphaticorgans but an increase in lymphocyte percentage. Treated animals showeda significant increase in the circulating lymphocytes at the early timepoints post-injection (15.11±2.81 vs. 12.5±2.41%, p<0.046 pst/pri).Control animals had lymphocyte values at the time points tested(p=0.32).

Example 10 GHRH or Biological Equivalent Treatment Shows BeneficialEffects in Old Healthy Dogs

FIG. 20 shows the changes in values associated with blood components inold healthy dogs injected with 1000 mcg of pSP-HV-GHRH plasmid. Manyblood component values were monitored two-weeks post injectionincluding: WBC, RBC, HGB, lym %, total protein, albumin, globulin, A/Gratio, cholesterol, BUN, Creatinine, phosphorous, Calcium, glucose. Olddogs treated with the pSP-HV-GHRH plasmid therapy, had increased RBChemoglobin and hematocrit levels two weeks post-injection compared withper-injected values. In addition, treated dogs have a significantdecrease in urea levels, increased total protein levels and normalglucose levels. An increase in Ca/P ratio is an indication of boneremodeling.

Example 11 Treatment of Anemia

It is well known that erythroid cell number is primarily regulated byerythropoietin (“EPO”) but is impacted by many growth factors. Forexample, hypophysectomized rats show low blood cell counts forerythroid, myeloid, and lymphoid cells, and there is extensiveliterature showing effects of both GH, and IGF-I on all hematopoieticlineages (Kurtz et al., 1990; Kurtz et al., 1982; Claustres et al.,1987). In polycytemia vera, patients present increased sensitivity oferythroid progenitor cells to IGF-I, elevated level of IGFBP-1 andconsequently overproduction of red blood cells (Mirza et al., 1997;Correa et al., 1994). There is evidence to support the concept thatIGF-I rather than EPO modulates erythropoiesis during accelerated growthor catabolism and thus manages a proportional increase in body mass andoxygen transport capacity (Kurtz et al., 1990). IGF-I is importantfactor regulating erythropoiesis in uremic patients (Urena et al.,1992). Moreover, the effect of treatment with recombinant human GH inanemic patients with panhypopituitarism is known. After the treatmentwith human GH plasma EPO levels double, with a concomitant increase ofHb concentration to normal levels. When the administration of human GHis interrupted, both plasma EPO levels and Hb concentrations decrease.

In injected dogs, a rapid correction of the anemia was obtained, asearly as two weeks after the plasmid injection. Red blood cells (RBC)increased by 8.9%, 9-27 days and the normal values were maintained to 56days post-injection (“pti”), compared with pre-injection (“pri”) values(6.27±0.33, vs. 5.75±0.39, p<0.027), while the control group had a 6%decrease in their RBC levels (6.00±0.2 vs. 5.5±0.2) (p<0.006 compared topost-injection controls) in the same period of time (FIG. 5). Hemoglobinlevels (FIG. 6) increased by 6.8% (14.68±0.78 vs. 13.74±0.97 g/lpti/pri), while the control group had a 5.7% decrease in the same periodof time (12.9±0.4 vs. 13.7±0.4 g/l), p<0.01 compared to post-injectioncontrols. Hematocrit levels (FIG. 7) increased significantly, by 8.26%(42.22±2.16 vs. 39±2.62%, p<0.032 pst/pri). In the same period, controlvalues decreased by 7.6% (36.9±1.4 vs. 39.9±1.6%, p<0.012 compared withpst).

In a pre-clinical study on dog cancer patients, a rapid correction ofthe anemia was obtained, as early as two weeks after the plasmidinjection. At the beginning of the study, the patients were in acatabolic state, with hemoglobin (Hb), hematocrit (“PVC”) and red bloodcell (“RBC”) values significantly lower than normal dogs. After theplasmid injection, the dogs entered a rapid reverse stage, and becamebiochemically anabolic, mimicking a rapid growth process, as in thestudy described previously (“Growth Hormone Axis and the immunefunction”) on young rats in the growth phase. Hb, PVC and RBC valuesincreased with 10-25%, values significant statistically, and normalizedtwo weeks after the beginning of the therapy. All values were within thenormal limits throughout the experiment. Although not wanting to bebound by theory, our hypothesis is that stimulation of the GHRH—GH—IGF-Iaxis in a catabolic state is stimulating erytropoiesis most probablythrough stimulation of erythropoietin. When the patients are reversed toa normal anabolic state, the natural GH effect is to induce a slightdegree of anemia. Nevertheless, these patients have cancer, and thenatural course of the disease is towards catabolism. Patients will bemaintained in balance by these contradictory mechanisms, thus the Hb,PVC and RBC values will be corrected to normal, but never exceed theupper normal limits, as shown in our studies.

Target gene expression also can be regulated by incorporating molecularswitch technology as schematically diagramed in FIG. 9. A commerciallyavailable system for ligand-dependent induction of transgene expressionhas a registered trademark name of GeneSwitch®. The GeneSwitch®technology is based on a C-terminally truncated progesterone receptorthat actually synthesized, but fails to bind to its natural agonist,progesterone. Instead the truncated progesterone receptor is onlyactivated by antiprogestins, such as mifepristone (“MFP”) (Vegeto etal., 1992; Xu et al., 1996). A similar system is used for the chimericregulator protein of the GeneSwitch™ system, which consists of theligand binding domain of the truncated human progesterone receptor thathas been fused to the DNA binding domain of the yeast GAL4 protein(which binds a specific 17 bp recognition sequence) and atranscriptional activation domain from the p65 subunit of human NF-KB(Abruzzese et al., 1999). The gene for the GeneSwitch regulator proteinwas inserted into a myogenic expression vector, designated pGS1633,which is expressed constitutively under the control of a muscle-specificskeletal alpha-actin (“SK”) promoter The GHRH plasmid, designated,p6xGal4/TATA-GHRH, or pGHRH1633 contains an inducible promoter thatconsists of six copies of the 17-mer Gal4 binding site fused to aminimal TATA box promoter. The GHRH coding sequence is a 228-bp fragmentof super-porcine mutated GHRH cDNA, termed HV-GHRH (Draghia-Akli et al.,1999). The HV-GHRH molecule displays a high degree of stability inserum, with a half-life of 6 hours, versus the natural GHRH, that has a6-12 minutes half-life. The muscle-specific GeneSwitch and inducibleGHRH plasmids both have a 5′ untranslated region that contains asynthetic intron, and a 3′ untranslated region/poly(A) site that is fromthe human GH gene.

Example 12 Pharmacological aand Toxicological Effects of Exogenous GHAdministration in Normal Animal Subjects

Because porcine GH (pGH) is structurally identical to canine GH, pGH wasused in different studies on dogs. In one of these studies, pGH wasadministered for a 14-weeks in dogs. Porcine GH caused increased bodyweight gain in mid- and high-dose groups (2.8 kg and 4.7 kg,respectively), compared to 0.4 kg and 0.8 kg in control and low-dosegroups, respectively. In pGH-treated dogs, increased skin thickness seengrossly correlated histologically with increased dermal collagen. Therewas no gross or histomorphological evidence of edema. There weredose-related increased serum IGF-I levels (approximately 2-10-fold) thatcorrelated with the elevated serum GH levels in pGH-treated dogs. Also,increased serum insulin levels through the mid dose were seen throughoutthe study. In high-dose dogs, the insulin levels remained elevated over24 hr postdose. The serum glucose levels in fasted dogs remained withinthe control range and there was no chronic hyperglycemia based onglycosylated hemoglobin levels. Renal glomerular changes, significantpolyuria with decreased urine specific gravity, and increased seruminsulin levels suggested that the dogs had early insulin-resistantdiabetes. There was minimal or no biologically significant effect of pGHon serum T3, T4, and cortisol levels in dogs. Other serum biochemicalchanges in pGH-treated dogs included decreased urea nitrogen andcreatinine, and increased potassium, cholesterol, and triglycerides.Significant increases in serum calcium and phosphorous levels andalkaline phosphatase activity (bone isozyme) correlated with thehistological changes in bone. In pGH-treated dogs, there was adose-related normochromic, normocytic, nonregenerative anemia. Thechanges described above, except for the anemia, are related to cataboliceffects of high doses of GH (Prahalada et al., 1998)

Example 13 Plasmid Mediated GHRH Delivery Slows Tumor Growth, PreventsKidney Failure and Increases Survivability in Tumor-Bearing Animals

Immunocompetent C57/B16 mice or immunocompromised nude mice wereinjected with tumor lines (C57/B16 mice were implanted with a Lewis lungrat adenocarcinoma line, while the nude mice were implanted with a humanlung small cell adenocarcinoma line). At 1 day after the tumorimplantation, mice were treated with either constitutively active GHRHconstruct, an inducible GHRH construct or with a β-galactosidaseexpressing construct (as negative control). Tumor established slower(FIG. 10 and FIG. 14), and developed less rapidly in the GHRH treatedanimals. Consequently, GHRH treated animals survived longer (FIG. 11),and were less likely to develop kidney failure (FIG. 12), than controls.Metastases are less likely to develop (FIG. 13).

The invention described herein involves the utilization of severaldistinctive GHRH or biological equivalent nucleic acid sequences. Basedupon the current understanding of protein-protein interactions, it isneither obvious nor possible to accurately speculate upon the in vivoparameters (e.g. half life, efficacy, post-translational modifications,etc.) of a GHRH sequence that contains a point mutation which alters asingle amino acid in the polypeptide chain. However, based on the knownart and the teachings of this specification, one skilled in the artwould know how to perform the plasmid mediated supplementationexperimentation(s), characterizing variations and permutations on anyunique nucleic acid sequence in a specific tissue to accurately evaluatethe in vivo effect on normal or chronic conditions within a livingorganism. Therefore, the utilization of the distinctive nucleic acidsequence encoding GHRH or biological equivalent thereof or correspondingrecombinant protein as a plasmid-mediated method to treat anemia;increase total red blood cell mass; reverse the wasting; reverseabnormal weight loss; treat immune dysfunction; reverse the suppressionof lymphopoesis; and/or extend life expectancy for a chronically illsubject could not have been predicted based on speculation.

Although not wanting to be bound by theory, it is believed that anincrease in GHRH will increase the GH levels sufficient to treat anemia;increase total red blood cell mass; reverse the wasting; reverseabnormal weight loss; treat immune dysfunction; reverse the suppressionof lymphopoesis; or extend life expectancy for the chronically illsubject. Hormones (e.g. GHRH and GH) often contain a complexfeedback-regulated pathway, which are further complicated by chronicconditions such as cancer or AIDS. Without direct experimentation ofGHRH or biological equivalents used in plasmid-mediated supplementationor the teachings provided herein, beneficial therapy could not have beenpredicted by one skilled in the art to determine which concentrations ofnon-native encoded sequences will yield desired results. Idealregulation of a nucleic acid sequence encoding GHRH or biologicalequivalent thereof is further complicated by the tissue used for plasmidmediated supplementation, and would not have been obvious to one skilledin the art without actual experimentations with the distinctive sequencein a particular tissue. The invention described herein contains thedescriptions and results of essential experimentation that exploredtissue specific and inducible regulation of distinctive nucleic acidsequences that encoded GHRH or biological equivalent thereof, which wasnot obvious based upon prior art. The present invention is a significantstep forward in developing non-viral therapy for large animals,including humans. In order for nucleic acid-based therapies to betransferred from rodents to large mammals, and ultimately to humans, itwas surprising that extremely low quantities of plasmid were effective.It is shown herein that as little as 0.2 mg plasmid delivered under theproper electroporation conditions had an important biological impactthat reversed wasting, increase weight gain, and extend life in anailing canine subject. This plasmid quantity was 100 fold lower than thetheoretical one, and could not have been predicted from the relativedoses used in rodents (in average 1 mg/kg).

The treatment of anemia, wasting, or immune dysfunction; the increase intotal red blood cell mass; the reverse of abnormal weight loss; thereverse in the suppression of lymphopoesis; and/or the extension of lifeexpectancy for a chronically ill subject are a consequence of the GHRHmolecules present in the subjects circulation, regardless of the meansof the delivery. For example, one would obtain the same effect bydelivering the appropriate quantities of GHRH or an analog thereof byclassical recombinant protein therapy or nucleic acid transfer.Accordingly, successful plasmid-mediated supplementation requiresaccurate delivery of encoded sequences to the cells of a subject thatresults in expression of the gene product at levels appropriate toproduce a biological effect. The duration of treatment will extendthrough the course of the disease symptoms, and possibly continuously.Since the method to deliver nucleic acid sequences to the cells of asubject is highly dependent on specific diseases and the encoded gene,it could not have been predicted by one skilled in the art which methodand conditions are appropriate without the teachings of thisspecification.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objectives and obtain the ends andadvantages mentioned as well as those inherent therein. Methods,procedures, techniques, and kits described herein are presentlyrepresentative of the preferred embodiments and are intended to beexemplary and are not intended as limitations of the scope. Changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention or defined by the scopeof the invention.

1. A method of treating anemia in a patient, the anemia being caused bya decrease in red blood cell production, comprising: (a) administeringinto the muscle of the subject an effective amount of a plasmidexpression construct encoding HV-GHRH (SEQ ID NO: 1), wherein theencoded HV-GHRH is operably linked to a SPc5-12 promoter of the plasmidexpression construct, and wherein the plasmid expression construct ismixed with a transfection-facilitating polypeptide; (b) electroporatingthe muscle having the effective amount of plasmid expression constructencoding the HV-GHRH (SEQ ID NO: 1), wherein the subject is adomesticated animal or human and wherein the HV-GHRH is biologicallyactive and expressed in the muscle cells and released by the musclecells causing a facilitation of growth hormone secretion and therebycausing increased red blood cell production, thereby treating theanemia.